Use and administration of bacterial efflux pump inhibitors

ABSTRACT

This invention provides for efflux pump inhibitors to be co-administered with antimicrobial agents for the treatment of infections caused by drug resistant pathogens, novel efflux pump inhibitors, combined dosage forms of efflux pump inhibitors with an antimicrobial, and novel medical methods.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of antimicrobial agents and more specifically it relates to the use of pentamidine and analogous compositions as efflux pump inhibitors to be co-administered with antimicrobial agents for the treatment of infections caused by drug resistant pathogens. The invention also includes novel compounds useful as efflux pump inhibitors and compositions and devices comprising an efflux pump inhibitor and an antimicrobial agent.

2. Description of the Related Art

Antibiotics have been effective tools in the treatment of infectious diseases during the last half-century. From the development of antibiotic therapy to the late 1980s there was almost complete control over bacterial infections in developed countries. However, in response to the pressure of antibiotic usage, multiple resistance mechanisms have become widespread and are threatening the clinical utility of antibacterial therapy. The increase in antibiotic resistant strains has been particularly common in major hospitals and care centers. The consequences of the increase in resistant strains include higher morbidity and mortality, longer patient hospitalization, and an increase in treatment costs.

Bacteria have developed several different mechanisms to overcome the action of antibiotics. These mechanisms of resistance can be specific for a molecule or a family of antibiotics, or can be non-specific and be involved in resistance to unrelated antibiotics. Several mechanisms of resistance can exist in a single bacterial strain, and those mechanisms may act independently or they may act synergistically to overcome the action of an antibiotic or a combination of antibiotics. Specific mechanisms include degradation of the drug, inactivation of the drug by enzymatic modification, and alteration of the drug target. There are, however, more general mechanisms of drug resistance, in which access of the antibiotic to the target is prevented or reduced by decreasing the transport of the antibiotic into the cell or by increasing the efflux of the drug from the cell to the outside medium. Both mechanisms can lower the concentration of drug at the target site and allow bacterial survival in the presence of one or more antibiotics that would otherwise inhibit or kill the bacterial cells. Some bacteria utilize both mechanisms, combining a low permeability of the cell wall (including membranes) with an active efflux of antibiotics.

In recent years interest in efflux-mediated resistance in bacteria has been triggered by the growing amount of data implicating efflux pumps in clinical isolates. The phenomenon of antibiotic efflux was first discovered in 1980, in the context of the mechanism of tetracycline resistance in enterobacteria. Since then, it has been shown that efflux of antibiotics can be mediated by more than one pump in a single organism, and that almost all antibiotics are subject to resistance by this mechanism.

Some efflux pumps selectively extrude specific antibiotics. Examples of such pumps include the Tet or CmlA transporters, which can extrude tetracycline or chloramphenicol, respectively. Other efflux pumps, so-called multi-drug resistance (MDR) pumps, extrude a variety of structurally diverse compounds. In the latter case, a single efflux system may confer resistance to multiple antibiotics with different modes of action. In this respect, bacterial MDR pumps are similar to mammalian MDR transporters. In fact, one such pump, P-glycoprotein, the first discovered MDR pump, confers multiple drug resistance on cancer cells and is considered to be one of the major reasons tumor resistance to anti-cancer therapy. A typical example of bacterial MDR pump is MexAB-OprM from Pseudomonas aeruginosa. This pump has been shown to affect the susceptibility of the organism to almost all antibiotic classes which fluoroquinolones, β-lactams, macrolides, phenicols, tetracyclines, and oxazolidinones.

Efflux pumps in gram-positive bacteria excrete their substrates across a single cytoplasmic membrane. This is also the case for some pumps in gram-negative bacteria, and as a result their substrates are effluxed into the periplasmic space. Other efflux pumps from gram-negative bacteria efflux their substrates directly into the external medium, bypassing the periplasm and the outer membrane. These pumps are organized in complex three component structures, which traverse both inner and outer membranes. They consist of a transporter located in the cytoplasmic membrane, an outer membrane channel and a periplasmic ‘linker’ protein, which brings the other two components into contact. It is clearly advantageous for gram-negative bacteria to efflux drugs by bypassing the periplasm and outer membrane. In gram-negative bacteria the outer membrane significantly slows down the entry of both lipophilic and hydrophilic agents. The former, such as erythromycin and fusidic acid, are hindered by the lipopolysaccharide components of the outer leaflet of the outer membrane bilayer. Hydrophilic agents cross the outer membrane through water-filled porins whose size prevents rapid diffusion, even for small compounds such as fluoroquinolones and some β-lactams. Thus, direct efflux creates the possibility for two different mechanisms to work synergistically to provide the cell with a potent defense mechanism. Furthermore, direct efflux into the medium leads to decreased amounts of drugs not only in the cytoplasmic but also in the periplasmic space. This could explain the apparently paradoxical finding that efflux pumps protect gram-negative bacteria from β-lactam antibiotics whose target penicillin-binding proteins are found in the periplasm.

Many MDR pumps are encoded by the genes, which are normal constituents of bacterial chromosomes In this case increased antibiotic resistance is a consequence of over-expression of these genes. Thus bacteria have the potential to develop multi-drug resistance without the acquisition of multiple specific resistance determinants. In some cases, the simultaneous operation of efflux pumps and other resistance mechanisms in the same cell results in synergistic effects.

While some genes encoding efflux pumps are not expressed in wild type cells and require induction or regulatory mutations for expression to occur, other efflux genes are expressed constitutively. As a result wild type cells have basal level of efflux activity. This basal activity of multi-drug efflux pumps in wild type cells contribute to intrinsic antibiotic resistance, or more properly, decreased antibiotic susceptibility. This intrinsic resistance may be low enough for the bacteria to still be clinically susceptible to therapy. However, the bacteria might be even more susceptible if efflux pumps were rendered non-functional, allowing lower doses of antibiotics to be effective. To illustrate, P. aeruginosa laboratory-derived mutant strain PAM1626, which does not produce any measurable amounts of efflux pump is 8 to 10 fold more susceptible to levofloxacin and meropenem than the parent strain P. aeruginosa PAM1020, which produces the basal level of MexAB-OprM efflux pump. Were it not for efflux pumps, the spectrum of activity of many so-called ‘gram-positive’ antibiotics could be expanded to previously non-susceptible gram-negative species. This can be applied to ‘narrow-spectrum’ β-lactams, macrolides, lincosamides, streptogramins, rifamycins, fusidic acid, and oxazolidinones—all of which have a potent antibacterial effect against engineered mutants lacking efflux pumps.

It is clear that in many cases, a dramatic effect on the susceptibility of problematic pathogens would be greatly enhanced if efflux-mediated resistance were to be nullified. Two approaches to combat the adverse effects of efflux on the efficacy of antimicrobial agents can be envisioned: identification of derivatives of known antibiotics that are not effluxed and development of therapeutic agents that inhibit transport activity of efflux pumps and could be used in combination with existing antibiotics to increase their potency.

There are several examples when the first approach has been successfully reduced to practice. These examples include new fluoroquinolones, which are not affected by multidrug resistance pumps in Staphylococcus aureus or Streptococcus pneumonia or new tetracycline and macrolide derivatives which are not recognized by the corresponding antibiotic-specific pumps. However, this approach appears to be much less successful in the case of multidrug resistance pumps from gram-negative bacteria. In gram-negative bacteria, particular restrictions are imposed on the structure of successful drugs: they must be amphiphilic in order to cross both membranes. It is this very property that makes antibiotics good substrates of multi-drug resistance efflux pumps from gram-negative bacteria. In the case of these bacteria the efflux pump inhibitory approach becomes the major strategy in improving the clinical effectiveness of existing antibacterial therapy.

The efflux pump inhibitory approach was first validated in the case of mammalian P-glycoprotein. And the first inhibitors have been found among compounds with previously described and quite variable pharmacological activities. For example, P-glycoprotein-mediated resistance, can be reversed by calcium channel blockers such as verpamyl and azidopine, immunosuppressive agents cyclosporin A and FK506 as well as antifungal agents such as rapamycin and FK520 (Raymond et al, 1994). It is important that efflux pump inhibitory activity was by no means connected to other activities of these compounds. In fact, the most advanced inhibitor of P-glycoprotein is a structural derivative of cyclosporin A and is devoid if immunosuppressive activity.

Improved compositions and methods for controlling drug resistance in microbes, in particular in microbes that are highly resistant to drugs, would be of tremendous benefit. The present invention provides such compositions and methods.

SUMMARY OF THE INVENTION

It has been discovered that pentamidine is capable of inhibiting multidrug-resistance pumps from various gram-negative bacteria. When administered to a patient suffering from a microbial infection that employs efflux pump(s) as a resistance mechanism, pentamidine inhibits the activity of the pump(s) allowing a co-administrated antimicrobial agent to accumulate in sufficient concentration to inhibit the microbe and treat the infection. Thus, in one aspect the present invention relates to a method for inhibiting a microbial infection that employs an efflux pump resistance mechanism, comprising contacting the cell with an efflux pump inhibitor optionally in combination with an antimicrobial agent. The efflux pump inhibitor may be pentamidine or a structurally related compound.

In a further related aspect, this invention includes a method for prophylactic treatment of a mammal. In this method, an efflux pump inhibitor is administered to a mammal at risk of a microbial infection, e.g., a bacterial infection. In some embodiments, an antimicrobial agent is administered in combination with or coadministered with the efflux pump inhibitor.

This invention also features a method of enhancing the antimicrobial activity of an antimicrobial agent against a microbe, in which such a microbe is contacted with an efflux pump inhibitor, and an antibacterial agent.

In a further aspect this invention provides pharmaceutical compositions effective for treatment of an infection of an animal, e.g., a mammal, by a microbe, such as a bacterium or a fungus. The composition includes a pharmaceutically acceptable carrier and an efflux pump inhibitor as described above. The invention also provides antimicrobial formulations that include an antimicrobial agent, an efflux pump inhibitor, and a carrier. In preferred embodiments, the antimicrobial agent is an antibacterial agent.

In a further aspect the efflux pump inhibitor is administered to the lungs as an aerosol. The antimicrobial agent may be administered in conjunction with the efflux pump inhibitor by any known means. Finally, the present invention includes not only a large number of known compounds that are now disclosed as useful as efflux pump inhibitors, but also includes a large number of novel compounds with that utility, and those new compounds comprise one aspect of the present invention.

Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

In one embodiment, a method is provided for treating a bacterial infection in a subject, including co-administering to a subject infected with a bacteria an antimicrobial agent and a compound of formula I in such a manner as to achieve an effective efflux pump inhibitory concentration of the compound of formula I at a site of infection:

wherein R₁ and R₂ are separately selected from the group consisting of hydrogen, methyl, amine, and C₁₋₄ alkylamine;

linkers L₁ and L₃ are separately selected from the group consisting of amine, C₁₋₂ alkyl, and C₁₋₂ alkylamine or are separately absent;

aromatic rings A₁ and A₂ are separately selected from the group consisting of

wherein Z₁-Z₄ are separately selected from the group consisting of C and N, with the proviso that aromaticity of the aromatic rings are maintained;

Z₁-Z₄ that are C are optionally substituted with C₁₋₄ alkyl, CH₂NH₂, halogen, methoxy, CH₂C(O)NMe₂, C(O)NH₂, C(O)NMe₂, SO₂Me, or SO₂NH₂;

Z₁-Z₄ that are N are optionally quatemized to form

Y₁, Y₃, and Y₄ are separately selected from the group consisting of CH, N, NH, S, and O and Y₂ and Y₅ are separately selected from the group consisting of C and N, with the proviso that aromaticity of the aromatic rings are maintained;

Y₁, Y₃, and Y₄ that are C are optionally substituted with halogen, methoxy, CH₂C(O)NH₂, CH₂C(O)NMe₂, C(O)NMe₂, SO₂Me, or SO₂NH₂;

Y₁, Y₃, and Y₄ that are N are optionally quaternized to form

wherein R₃ is C₁₋₄ alkyl, CH₂C(O)NH₂, or CH₂C(O)NMe₂;

linker L₂ is a 1 to 12 unit chain optionally containing units selected from the group consisting of CH₂, C(CH₃)₂, O, C(O), S, S(O), S(O)₂, NH, NR₄, ═N—, phenyl, monocyclic 5-membered heteroaryl, monocyclic 6-membered heteroaryl, —CH═CH— cis, —CH═CH— trans, NHC(O)NH, NR₄C(O)NH, NHC(O)NR₄, NR₄C(O)NR₄, OC(O)NH, NR₄C(O)O, OC(O)NR₄, and NHC(O)O with the proviso that L₂ does not contain a C(O)NH, C(O)NR₄, C(O)O, or C(O)S unit;

wherein the 5-membered heteroaryls are selected from the group consisting of imidazole, furane, thiophene, thiazole, isothiazole, oxazole, isoxazole, 1,2,3-oxadiazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, 1,2,3-triazole, and 1,3,4-triazole;

the 6-membered heteroaryls are selected from the group consisting of pyridine, pyrimidine, pyridazine, 1,2,4-triazine, and 1,3,5-triazine; and

R₄ is selected from the group consisting of H and C₁₋₄ alkyl.

In some embodiment, the method further includes identifying the subject as a subject infected with a bacteria that is resistant to the antimicrobial agent. In some embodiments, the method further includes identifying the subject as a subject infected with a bacteria that is capable of developing resistance to the antimicrobial agent. In some embodiments, the resistance is at least partly efflux pump-mediated. In some embodiments, the efflux pump inhibitory concentration is sufficient to overcome or suppress the emergence of efflux pump-mediated resistance in the bacteria.

In another embodiment, a method is provided for prophylactic treatment of a subject, including co-administering to a subject at risk of infection with a bacteria an antimicrobial agent and a compound of formula I in such a manner as to achieve an efflux pump inhibitory concentration of the compound of formula I as described above at a potential site of infection. In one embodiment, the method further includes identifying the subject as a subject at risk of the bacterial infection.

In another embodiment, a method is provided for treating a microbial infection in a subject, including administering to a subject infected with a microbe a compound of formula I as described above in such a manner as to achieve an efflux pump inhibitory concentration of the compound of formula I at a site of infection, with the proviso that the compound of formula I does not include pentamidine, propamindine, hexamidine, dibromopropamidine, phenamidine, amicarbalide, diaminazene, and stilbamidine. In one embodiment, the method further comprising identifying the subject as a subject infected with a microbe that is resistant to an antimicrobial agent. In one embodiment, the method further includes identifying the subject as a subject infected with a microbe that is capable of developing resistance to an antimicrobial agent. In one embodiment, the microbe is a bacteria. One embodiment further includes co-administering with the compound of formula I an antimicrobial agent. In one embodiment, the antimicrobial agent is an antibacterial agent. In one embodiment, the MIC of the the compound of formula I for the microbe is greater than about 32 μg/ml.

In another embodiment, a method is provided for prophylactic treatment of a subject, comprising administering to a subject at risk of infection with a microbe a compound of formula I as described above in such a manner as to achieve an efflux pump inhibitory concentration of the compound of formula I at a site of infection, with the proviso that the compound of formula I does not include pentamidine, propamindine, hexamidine, dibromopropamidine, phenamidine, amicarbalide, diaminazene, and stilbamidine. In one embodiment, the method further includes identifying the subject as a subject at risk of the microbial infection. In one embodiment, the microbe is a bacteria. In one embodiment, the method further includes co-administering with the compound of formula I an antimicrobial agent. In one embodiment, the antimicrobial agent is an antibacterial agent. In one embodiment, the MIC of the the compound of formula I for the microbe is greater than about 32 μg/ml.

In another embodiment, a method is provided for administering a compound of formula I as described above, including providing for inhalation of an aerosol comprising the compound of formula I, the aerosol having a mean particle size from about 2 microns to about 4 microns mass median aerodynamic diameter and a particle size geometric standard deviation of less than or equal to about 2 microns.

In some embodiments of the methods described above, an effective efflux pump inhibitory concentration of the compound of formula I remains at the site of infection for at least about a 2 hour period. In some embodiments of the methods described above, an effective efflux pump inhibitory concentration of the compound of formula I remains at the site of infection for at least about 4 hour period. In some embodiments of the methods described above, an effective efflux pump inhibitory concentration of the compound of formula I is at the site of infection at a same time as the antimicrobial agent is present at its peak period. In some embodiments of the methods described above, an effective efflux pump inhibitory concentration of the compound of formula I is at the site of infection for at least about 25% of the time the antimicrobial agent is present at its peak period. In some embodiments of the methods described above, the effective efflux pump inhibitory concentration is sufficient to improve an AUC/MIC ratio of the antimicrobial agent by at least about 25%. In some embodiments of the methods described above, the effective efflux pump inhibitory concentration is sufficient to improve time-above-MIC for the antimicrobial agent by at least about 25%. In some embodiments of the methods described above, the effective efflux pump inhibitory concentration is sufficient to cause a therapeutic effect. In some embodiments of the methods described above, the site of infection is localized. In some embodiments of the methods described above, the site of infection is a pulmonary site. In some embodiments of the methods described above, the bacteria is a Pseudomonas. In some embodiments of the methods described above, the compound of formula I is administered as an aerosol. In some embodiments of the methods described above, the compound of formula I is administered in one or more administrations so as to achieve a respirable delivered dose daily of at least about 15 mg. In some embodiments of the methods described above, the compound of formula I is administered in one or more administrations so as to achieve a respirable delivered dose daily of at least about 40 mg. In some embodiments of the methods described above, the compound of formula I is administered in one or more administrations so as to achieve a respirable delivered dose daily of at least about 60 mg. In some embodiments of the methods described above, the administering step comprises administering the antimicrobial agent and the compound of formula I in a predetermined ratio. In some embodiments of the methods described above, the administering step comprises administering the antimicrobial agent and the compound of formula I by separate routes of administration. In some embodiments of the methods described above, the antimicrobial agent and the compound of formula I are administered sequentially. In some embodiments of the methods described above, the antimicrobial agent and the compound of formula I are administered simultaneously. In some embodiments of the methods described above, the antimicrobial agent and the compound of formula I are administered in a combined, fixed dosage form. In some embodiments of the methods described above, the antimicrobial agent is a substrate of an efflux pump in the bacteria. In some embodiments of the methods described above, the antimicrobial agent is an antibacterial agent. In some embodiments of the methods described above, the antimicrobial agent is a quinolone. In some embodiments of the methods described above, the antimicrobial agent is a fluoroquinolone. In some embodiments of the methods described above, the antimicrobial agent is selected from the group consisting of ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, pefloxacin, sparfloxacin, garenoxacin, sitafloxacin, and DX-619. In some embodiments of the methods described above, the antimicrobial agent is selected from the group consisting of an aminoglycoside, beta-lactam, coumermycin, chloramphenical, daptomycin, glycopeptide, glycylcycline, ketolide, macrolide, oxazolidonone, rifamycin, stroptogramin and tetracycline. In some embodiments of the methods described above, the bacteria is a gram-negative bacteria. In some embodiments of the methods described above, the bacteria is selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, and Bacteroides splanchnicus. In some embodiments of the methods described above, the subject is a human. In some embodiments of the methods described above, the subject is a human with cystic fibrosis. In some embodiments of the methods described above, the subject is a human with pneumonia, a chronic obstructive pulmonary disease, or sinusitis, or a human being mechanically ventilatated.

In another embodiment, a pharmaceutical product is provided including a fixed combination of an antimicrobial agent and a compound of formula I as described above. In one such embodiment, the antimicrobial and the compound of formula I are physically combined. In one embodiment, the antimicrobial and the compound of formula I are packaged together in separate containers. In one embodiment, the antimicrobial agent is a substrate of an efflux pump in a bacteria. In one embodiment, the antimicrobial is an antimicrobial used to treat infections caused by a gram negative bacteria. In one embodiment, the antimicrobial is an antimicrobial used to treat infections caused by one or more bacteria selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, and Bacteroides splanchnicus. In one embodiment, the antimicrobial is an antimicrobial used to treat infections caused by a bacteria that comprises an efflux pump that is inhibited by the compound of formula I. In one embodiment, the antimicrobial and the compound of formula I have a molar ratio equal to or greater than about 1 part compound of formula I to about 1 part antimicrobial.

In another embodiment, a pharmaceutical composition is provided that includes a solution of a compound of formula I as described above having an osmolality from about 200 mOsmol/kg to about 500 mOsmol/kg. In one such embodiment, the solution has a permeant ion concentration from about 50 mM to about 250 mM.

In another embodiment, a pharmaceutical composition is provided that includes a solution of a compound of formula I having a permeant ion concentration from about 50 mM to about 250 mM. In one such embodiment, one or more permeant ions in the composition are selected from the group consisting of chloride and bromide.

In another embodiment, system is provided for administering a compound of formula I that includes a container comprising a solution of a compound of formula I and a nebulizer coupled to the container and adapted to produce an aerosol of the solution having a mean particle size from about 2 microns to about 4 microns and a particle size geometric standard deviation of less than or equal to about 2 microns.

In one embodiment of the methods, products, systems, and compositions described above, R₁ and R₂ in the compound of formula I are amine groups. In one embodiment of the methods, products, systems, and compositions described above, L₂ in the compound of formula I is a 1 to 12 unit chain containing units selected from the group consisting of —CH₂—, —C(CH₃)₂—, —O—, —C(O)—, —S—, —S(O)—, —CH═CH— cis, and —CH═CH— trans. In one embodiment of the methods, products, systems, and compositions described above, the compound of formula I is selected from the group consisting of pentamidine, propamidine, hexamidine, dibromopropamidine,

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the effect of pentamidine on uptake of Leu-Nap in P. aeruginosa PAM 1723 cells. FIG. 1B illustrates the effect in PAM 1626 cells.

FIGS. 2A and 2B illustrate the effect of Leu-Nap concentration (120 μg/ml in FIG. 2B; 60 μg/ml in FIG. 2A) of the pentamidine inhibitory activity.

FIGS. 3A and 3B depict the results of the investigation of the outer membrane permeabilizing activity of PMBN (FIG. 3A) and pentamidine (FIG. 3B).

FIG. 4 depicts the results of the investigation of the outer membrane permeabilizing activity of pentamidine in the presence of Mg²⁺.

FIGS. 5A and 5B show the effect of CCCP (FIG. 5A) and pentamidine (FIG. 5B) on efflux of EtBr in P. aeruginosa.

FIG. 6 shows the susceptibility of the cystic fibrosis isolates P. aeruginosa to various antibiotics and fluoroquinolone/pentamidine combinations.

FIG. 7 depicts the impact of pentamidine on fluoroquinolone susceptibility of strains of P. aeruginosa isolated from patients with Cystic Fibrosis.

FIG. 8 depicts the impact of pentamidine on levofloxacin susceptibility of strains of P. aeruginosa isolated from patients with Cystic Fibrosis.

FIG. 9A depicts the impact of pentamidine alone on bacterial killing

FIG. 9B depicts the impact of pentamidine on bacterial killing by Levofloxacin.

FIG. 10A depicts inhibition Leu-Nap efflux from PAM1723 by Propamidine.

FIG. 10B depicts inhibition Leu-Nap efflux from PAM1626 by Propamidine.

FIG. 10C depicts inhibition Leu-Nap efflux from PAM1723 by Dibromopropamidine.

FIG. 10D depicts inhibition Leu-Nap efflux from PAM1626 by Dibromopropamidine.

FIG. 10E depicts inhibition Leu-Nap efflux from PAM1723 by Hexamidine.

FIG. 10F depicts inhibition Leu-Nap efflux from PAM1626 by Hexamidine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions

The term “administration” or “administering” refers to a method of giving a dosage of an antimicrobial pharmaceutical composition to a vertebrate or invertebrate, including a mammal, a bird, a fish, or an amphibian, where the method is, e.g., intrarespiratory, topical, oral, intravenous, intraperitoneal, or intramuscular. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the potential or actual bacterial infection, the microbe involved, and the severity of an actual microbial infection.

A “carrier” or “excipient” is a compound or material used to facilitate administration of the compound, for example, to increase the solubility of the compound. Solid carriers include, e.g., starch, lactose, dicalcium phosphate, sucrose, and kaolin. Liquid carriers include, e.g., sterile water, saline, buffers, non-ionic surfactants, and edible oils such as oil, peanut and sesame oils. In addition, various adjuvants such as are commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, N.J. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press.

A “diagnostic” as used herein is a compound, method, system, or device that assists in the identification and characterization of a health or disease state. The diagnostic can be used in standard assays as is known in the art.

The term “efflux pump” refers to a protein assembly that exports substrate molecules from the cytoplasm or periplasm of a cell, in an energy dependent fashion. Thus an efflux pump will typically be located in the cytoplasmic membrane of the cell (spanning the cytoplasmic membrane). In Gram-negative bacteria the pump may span the periplasmic space and there may also be portion of the efflux pump, which spans the outer membrane.

An “efflux pump inhibitor” (“EPI”) is a compound that specifically interferes with the ability of an efflux pump to export its normal substrate, or other compounds such as an antibiotic. The inhibitor may have intrinsic antimicrobial (e.g., antibacterial) activity of its own, but at least a significant portion of the relevant activity is due to the efflux pump inhibiting activity.

“High throughput screening” as used herein refers to an assay that provides for multiple candidate agents or samples to be screened simultaneously. As further described below, examples of such assays may include the use of microtiter plates which are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.

The term “mammal” is used in its usual biological sense. Thus, it specifically includes humans, cattle, horses, dogs, and cats, but also includes many other species.

The term “microbial infection” refers to the invasion of the host organism, whether it be a vertebrate, invertebrate, fish, plant, bird, or mammal by pathogenic microbes. This includes the excessive growth of microbes that are normally present in or on the body of a mammal or other organism. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host mammal. Thus, a mammal is “suffering” from a microbial infection when excessive numbers of a microbial population are present in or on a mammal's body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of a mammal. Specifically, this description applies to a bacterial infection. Note that the present invention is also useful in treating microbial growth or contamination of cell cultures or other media, or inanimate surfaces or objects, and nothing herein should limit the present invention only to treatment of higher organisms, except when explicitly so specified in the claims.

The term “multidrug resistance pump” refers to an efflux pump that is not highly specific to a particular antibiotic. The term thus includes broad substrate pumps (efflux a number of compounds with varying structural characteristics). These pumps are different from pumps, which are highly specific individual antibiotics. Examples include tetracycline-specific efflux pumps, chloramphenicol-specific efflux pumps or macrolide-specific efflux pumps. Such efflux pumps are involved in resistance to specific antibiotics in bacteria. However, they do not confer resistance to other antibiotics. The genes for the specific pump components are found in plasmids in Gram-negative as well as in Gram-positive bacteria.

The term “pentamidine efflux pump inhibitor” refers to pentamidine, a metabolite of pentamidine, or a combination of pentamidine and one or more of its metabolites, including single steroisomers, mixtures of steroisomers and the pharmaceutically acceptable salts, and solvates thereof.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The term “pharmaceutically acceptable salt” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which are not biologically or otherwise undesirable. In many cases, the compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

“Solvate” refers to the compound formed by the interaction of a solvent and pentamidine, a metabolite, or salt thereof. Suitable solvates are pharmaceutically acceptable solvates including hydrates.

“Subject” as used herein, means a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.

In the context of the response of a microbe, such as a bacterium, to an antimicrobial agent, the term “susceptibility” refers to the sensitivity of the microbe for the presence of the antimicrobial agent. So, to increase the susceptibility means that the microbe will be inhibited by a lower concentration of the antimicrobial agent in the medium surrounding the microbial cells. This is equivalent to saying that the microbe is more sensitive to the antimicrobial agent. In most cases the minimum inhibitory concentration (MIC) of that antimicrobial agent will have been reduced.

By “therapeutically effective amount” or “pharmaceutically effective amount” is meant an amount of an efflux pump inhibitor, or amounts individually of an efflux pump inhibitor and an antimicrobial agent, as disclosed for this invention, which have a therapeutic effect. The doses of efflux pump inhibitor and antimicrobial agent which are useful in combination as a treatment are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount means those amounts of efflux pump inhibitor and antimicrobial agent which, when used in combination, produce the desired therapeutic effect as judged by clinical trial results and/or model animal infection studies. In particular embodiments, the efflux pump inhibitor and antimicrobial agent are combined in pre-determined proportions and thus a therapeutically effective amount would be an amount of the combination. This amount and the amount of the efflux pump inhibitor and antimicrobial agent individually can be routinely determined by one of skill in the art, and will vary, depending on several factors, such as the particular microbial strain involved and the particular efflux pump inhibitor and antimicrobial agent used. This amount can further depend upon the patient's height, weight, sex, age and medical history. For prophylactic treatments, a therapeutically effective amount is that amount which would be effective to prevent a microbial infection.

A “therapeutic effect” relieves, to some extent, one or more of the symptoms of the infection, and includes curing an infection. “Curing” means that the symptoms of active infection are eliminated, including the elimination of excessive members of viable microbe of those involved in the infection. However, certain long-term or permanent effects of the infection may exist even after a cure is obtained (such as extensive tissue damage).

“Treat,” “treatment,” or “treating,” as used herein refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. The term “prophylactic treatment” refers to treating a patient who is not yet infected, but who is susceptible to, or otherwise at risk of, a particular infection. The term “therapeutic treatment” refers to administering treatment to a patient already suffering from an infection. Thus, in preferred embodiments, treating is the administration to a mammal (either for therapeutic or prophylactic purposes) of therapeutically effective amounts of a pentamidine and an antibacterial (or antimicrobial) agent in combination (either simultaneously or serially).

As used herein, an “effective efflux pump inhibitory concentration” refers to the minimal concentration of the efflux pump inhibitor sufficient to achieve at least 25% of either maximum biochemical effect (MBE) or maximum potentiating effect (MPE) in vitro. In some embodiments, an effective efflux pump inhibitory concentration sufficient to achieve at least a 25%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of either maximum biochemical effect (MBE) or maximum potentiating effect (MPE) in vitro is provided. The theoretical maximum effect that an efflux pump inhibitor can have on a microorganism is a reduction in the level of activity of the pump to a level that is equivalent to that observed in otherwise identical strains that lack all of the efflux pumps for a particular substrate. As used herein, the “MBE” is the maximum measurable biochemical effect an inhibitor can have on the efflux pump activity for a substrate. This level can be equal to or less than the theoretical maximum effect. As used herein, the “MPE” is the maximum potentiating effect that an efflux pump inhibitor can have on activity of an antimicrobial agent against the microorganism. A potentiating effect is the increase in susceptibility of a microorganism to an antimicrobial agent in the presence of an efflux pump inhibitor. The MPE can be equal to or less than the theoretical maximum potentiating effect, which is the ratio in antimicrobial susceptibility of otherwise identical strains that either possess or lack all of the efflux pumps for the antimicrobial agent.

MBE may be determined in whole-cell uptake experiments. The rate of uptake of an efflux pump substrate (antibacterial agent or non-antibiotic substrate) into intact cells of each type may be determined. To determine the rate of uptake, cells may be incubated with an efflux pump substrate, equal samples may be removed at different times and the amount of the substrate inside cells may be determined at each time point. The rate of uptake is defined as an amount of the substrate accumulated inside cells per time unit. This determination of intracellular concentration is facilitated if the substrate is fluorescent or radioactive. Otherwise, substrate can be extracted out of cells and its amount determined using HPLC. Alternatively, the rate of uptake can be monitored continuously in real time if the intracellular and extracellular substrate has different fluorescence or absorbance. One example is ethidium bromide, which upon entry inside cells binds to DNA and becomes intensely fluorescent. Another example is Leu-β-naphtylamide, which is not fluorescent in solution but upon entry into cells undergoes hydrolysis and produces highly fluorescent β-naphtylamine.

The rate of uptake is affected by both the rate of entry inside the cell and the rate of efflux. Since the rate of entry is the same for both cell types, the difference in uptake rates reflects the difference in efflux activity. The ratio between the rates of antibiotic uptake of two strains is determined and defined as a maximum biochemical effect (MBE).

The MPE may be determined in a standard checkerboard assay (e.g., Antimicrobial Combinations. In Antibiotics in Laboratory Medicine, Ed. Victor Lorian, M.D., Fourth edition, 1996, pp 333-338) using broth microdilution method as recommended by the NCCLS (National Committee for Clinical Laboratory Standards (NCCLS))). In this assay, multiple dilutions of two drugs, namely an antibiotic and efflux pump inhibitor, may be tested, alone and in combination for their ability to inhibit bacterial growth. As a result, MIC of the pump expressing strain (defined as the lowest concentration of antibiotics, within the combination, at which the visible growth of the organism is completely inhibited) is determined in the presence of each concentration of inhibitor. Increasing concentrations of inhibitor result in a reduction of MIC determined without inhibitor until the maximum antibiotic potentiating effect is achieved. Ratio of MIC without inhibitor and in the presence of inhibitor concentration, which achieves maximum reduction in MIC may be determined and is defined as MPE.

In some embodiments, an effective efflux inhibitory concentration is high enough to elicit a therapeutic effect when an efflux pump inhibitor is combined with an antimicrobial agent. As used herein, a “therapeutic effect” is defined as a statistically significant reduction in bacterial load in a host, emergence of resistance, or improvement in infection symptoms as measured by human clinical results or animal studies.

Pharmacokinetics (PK) is concerned with the time course of antimicrobial concentration in the body. Pharmacodynamics (PD) is concerned with the relationship between pharmacokinetics and the antibiotic efficacy in vivo. PK/PD parameters correlate antibiotic exposure with antibiotic activity. The rate of killing by antibiotic is dependent on antibiotic mode of action and is determined by either the length of time necessary to kill (time-dependent) or the effect of increasing concentrations (concentration-dependent). Accordingly, to predict the therapeutic efficacy of antibiotics with diverse mechanisms of action different PK/PD parameters may be used.

“AUC/MIC ratio” is one example of a PK/PD parameter. AUC is defined as the area under the plasma or site-of-infection concentration-time curve of an antimicrobial agent in vivo (in animal or human). AUC/MIC ratio is determined by dividing the 24-hour-AUC for an individual antimicrobial by the MIC for the same antimicrobial determined in vitro. Activity of antibiotics with the dose-dependent killing (such as fluoroquinolones) is well predicted by the magnitude of the AUC/MIC ratio.

“Time above MIC” (T>MIC) is another PK/PD parameter. It is expressed a percentage of a dosage interval in which the plasma or site-of-infection level exceeds the MIC. Activity of antibiotics with the time-dependent killing (such as beta-lactams or oxazolidinones) is well predicted by the magnitude of the AUC/MIC ratio.

The term “dosing interval” refers to the time between administrations of the two sequential doses of a pharmaceutical's during multiple dosing regimens. For example, in the case of ciprofloxacin, which is administered twice daily (traditional regimen of 400 mg b.i.d) and levofloxacin, which is administered once a day (500 mg or 750 mg q.d.), the dosing intervals are 12 hours and 24 hours, respectively.

As used herein, the “peak period” of a pharmaceutical's in vivo concentration is defined as that time of the pharmaceutical dosing interval when the pharmaceutical concentration is not less than 50% of its maximum plasma or site-of-infection concentration. In some embodiments, “peak period” is used to describe an interval of antimicrobial dosing.

The “respirable delivered dose” is the amount of drug inhaled during the inspriatory phase of the simulator that is equal to or less than 5 microns using a breath simulator programmed to the European Standard pattern of: 15 breaths per minute, with an inspiration to expiration ratio of 1:1.

Methods of Efflux Pump Inhibition

In one embodiment, methods are provided for treating microbial infections by administering one or more efflux pump inhibitors disclosed herein. The efflux pump inhibitors may be administered in such a manner that an effective efflux pump inhibitory concentration of the efflux pump inhibitor is achieved at a site of infection. In some embodiments, an antimicrobial agent is co-administered with the efflux pump inhibitor. By “co-administer” it is meant that the efflux pump inhibitor is administered to a patient such that the efflux pump inhibitor as well as the co-administered compound may be found in the patient's bloodstream at the same time and/or at the site of infection at the same time, regardless of when the compounds are actually administered, including simultaneously or sequentially. In one advantageous embodiment, the pharmacokinetics of the efflux pump inhibitor and a co-administered antimicrobial agent are substantially the same. In some advantageous embodiments, the efflux pump inhibitor concentration at a site of infection inhibits the efflux pump of the microbial to an extent such that the co-administered antimicrobial agent has an increased intracellular concentration within the microbial than it would have if the efflux pump inhibitor were not present. Thus, in some embodiments, the effective MIC of the anti-microbial is decreased by the presence of the efflux pump inhibitor.

In some embodiments, efflux pump inhibitors disclosed herein are used to prevent infection with a microbial by administering the inhibitor to a subject at risk of infection with the microbial. In such cases, the efflux pump inhibitors may be administered in such a manner that an efflux pump inhibitory concentration of the efflux pump inhibitor is achieved at a potential site of infection.

In some embodiments, an effective efflux pump inhibitory concentration is maintained at a site of infection for at least about a 1 hour period, a 2 hour period, a 3 hour period, a 4 hour period, a 5 hour period, a 6 hour period or during the entire antimicrobial agent's dosing interval. In some embodiments, an effective efflux pump inhibitor concentration is present at a site of infection at the same time that an antimicrobial agent is present at its peak period. In some embodiments, the entire period during which an effective efflux pump inhibitory concentration exists at a site of infection during an efflux pump inhibitor dosing interval is contained within an antimicrobial agent's peak period. In other embodiments, at least about 25%, 50%, or 75% of the entire period during which an effective efflux pump inhibitory concentration exists at a site of infection during an efflux pump inhibitor dosing interval is contained within an antimicrobial agent's peak period.

In some embodiments, the microbial is a bacteria and the antimicrobial agent is an antibacterial agent. In some embodiments, the patient is not otherwise in need of pentamidine therapy; i.e., is not suffering from a condition for which pentamidine therapy is approved or commonly administered. In most cases, this means that the patient is not an immunocompromised patient, or is not infected with pneumocystis carnii or does not have pneumocystis carnii pneumonia. In other embodiments, the patient is further not suffering from leishmaniasis or trypanosomiasis.

Pentamidine

Pentamidine is currently used for the treatment of Pneumocystis carinii, Leishmania donovani, Trypanosoma brucei, T. gambiense, and T. rhodesiense infections. The structure of pentamidine is

It is commercially available and formulated for injection or inhalation. For injection, pentamidine is packaged as a nonpyrogenic, lyophilized product. After reconstitution, it is administered by intramuscular or intravenous injection.

Pentamidine is formulated as the isethionate salt, which is a white, crystalline powder soluble in water and glycerin and insoluble in ether, acetone, and chloroform. It is chemically designated 4,4′-diamidino-diphenoxypentane di(β-hydroxyethanesulfonate). The molecular formula is C₂₃H₃₆N₄O₁₀S₂ and the molecular weight is 592.68.

The antimicrobial mode of action of pentamidine is not fully understood. In vitro studies with mammalian tissues and the protozoan Crithidia oncopelti indicate that the drug interferes with nuclear metabolism, producing inhibition of the synthesis of DNA, RNA, phospholipids, and proteins.

In vitro inhibitory activity of pentamidine against Pneumocystis carini, is 0.3 μg/ml (“Highly active anti-Pneumocystis carinii compounds in a library of novel piperazine-linked bisbenzamidines and related compounds”, Cushion et al., Antimicrob. Agents Chemother., 48(11): 4209-16, 2004; “Novel bisbenzamidines as potential drug candidates for the treatment of Pneumocystis carinii pneumonia”, Vanden Eynde et al., Bioorg. Med. Chem. Lett., 14(17): 4545-8, 2004, both of which are incorporated herein by reference in their entirety). When pentamidine is administered as a slow IV infusion (1-2 hours) at a daily dose of 3 mg/kg, the peak serum concentration ranges from 0.5 to 3.4 μg/ml (“Effect of Gender and Race on the Pharmacokinetics of Pentamidine in HIV-Infected Patients”, Conte et al., Clinical Drug Investigation, 17 (4): 293-299, 1999, which is incorporated herein by reference in its entirety). Plasma levels decrease rapidly during the first two hours following an intravenous infusion of pentamidine isethionate to one-twentieth of peak levels, followed by a much slower decline. After 3 weeks of daily administration of 3 mg/kg dose of pentamidine, trough plasma concentration reached 61.1+/−56.0 ng/mL (“Intravenous or inhaled pentamidine for treating Pneumocystis carinii pneumonia in AIDS. A randomized trial”, Conte et al., Ann. Intern. Med., 113(3): 203-9, 1990, which is incorporated herein by reference in its entirety). In seven patients treated with daily i.m. doses of pentamidine at 4 mg/kg for 10 to 12 days, plasma concentrations were between 0.3 and 0.5 μg/mL. The patients continued to excrete decreasing amounts of pentamidine in urine up to six to eight weeks after cessation of the treatment. Systemic absorption during aerosolized therapy is minimal: peak plasma concentrations are found to be less than 5% than that observed following equivalent intravenous administration. Accumulation in the plasma does not occur with repeated inhalation as has been described with multiple intravenous dosing (“Concentrations of aerosolized pentamidine in bronchoalveolar lavage, systemic absorption, and excretion”, Conte et al., Antimicrob Agents Chemother. 32(10):1490-3, 1988, which is incorporated herein by reference in its entirety).

Higher pulmonary concentrations of pentamidine are observed during aerosol administration. Specifically, 24 hours after administration of 300 mg in a jet nebulizer, pentamidine concentration in bronchial alveolar lavage fluid supernatant and sediment was 23.2+/−7.75 ng/ml and 705+/−242 ng/ml, respectively (“Selective delivery of pentamidine to the lung by aerosol”, Montgomery et al., Am. Rev. Respir. Dis., 137(2):477-8, 1988, which is incorporated herein by reference in its entirety). The currently approved pentamidine aerosol delivery device (300 mg in a Respirgard II nebulizer) provides a mean total pulmonary deposition of nebulized pentamidine of 15.3 mg, which is 5.1% of the initial nebulizer dose (“Disposition of nebulized pentamidine measured using the direct radiolabel 123I-iodopentamidine”, O'Doherty et al., Nucl. Med. Commun., 14(1):8-11, 1993, which is incorporated herein by reference in its entirety). Due to the particle size created by the Respirgard II nebulizer, it is believed that the delivery of aerosolized pentamidine is mostly in the alveoli.

Surprisingly, it has been discovered that pentamidine is an efflux pump inhibitor and thus can be used in accordance with the methods described herein. Notably, it is has been discovered that the efflux pump inhibitory concentration of pentamidine, is greater than the in vivo concentration of pentamidine produced by currently approved delivery methods. Specifically, concentrations of 10 μg/ml to 20 μg/ml pentamidine has been shown effective to potentiate levofloxacin against resistant bacteria in vitro. Acordingly, in one embodiment, a novel method is provided that includes co-administering pentamidine and an antimicrobial agent in such a manner as to provide an efflux pump inhibitory concentration at a site of infection in order to enhance the efficacy of the antimicrobial agent.

In some embodiments, pentamidine metabolites are provided for use as efflux pump inhibitors. Pentamidine is rapidly metabolized in the body to at least seven primary metabolites. Some of these metabolites share one or more activities with pentamidine.

Seven pentamidine metabolites are shown below.

Pentamidine Analogs

Compounds that are structurally related to pentamidine may also be used as described herein. In some embodiments, these compounds have the structure of formula (I):

wherein R₁ and R₂ are separately selected from the group consisting of hydrogen, methyl, amine, and C₁₋₄ alkylamine; linkers L₁ and L₃ are separately selected from the group consisting of amine, C₁₋₂ alkyl, and C₁₋₂ alkylamine or are separately absent; aromatic rings A₁ and A₂ are separately selected from the group consisting of

-   Z₁-Z₄ are separately selected from the group consisting of C and N,     with the proviso that aromaticity of the aromatic rings are     maintained; Z₁-Z₄ that are C are optionally substituted with C₁₋₄     alkyl, CH₂NH₂, halogen, methoxy, CH₂C(O)NMe₂, C(O)NH₂, C(O)NMe₂,     SO₂Me, or SO₂NH₂; Z₁-Z₄ that are N are optionally quatemized to form

-   Y₁, Y₃, and Y₄ are separately selected from the group consisting of     CH, N, NH, S, and O and Y₂ and Y₅ are separately selected from the     group consisting of C and N, with the proviso that aromaticity of     the aromatic rings are maintained; Y₁, Y₃, and Y₄ that are C are     optionally substituted with halogen, methoxy, CH₂C(O)NH₂,     CH₂C(O)NMe₂, C(O)NMe₂, SO₂Me, or SO₂NH₂; Y₁, Y₃, and Y₄ that are N     are optionally quaternized to form

-   R₃ is C₁₋₄ alkyl, CH₂C(O)NH₂, or CH₂C(O)NMe₂; linker L₂ is a 1 to 12     unit chain optionally containing units selected from the group     consisting of CH₂, C(CH₃)₂, O, C(O), S, S(O), S(O)₂, NH, NR₄,     phenyl, monocyclic 5-membered heteroaryl, monocyclic 6-membered     heteroaryl, —CH═CH— cis, —CH═CH— trans, NHC(O)NH, NR₄C(O)NH,     NHC(O)NR₄, NR₄C(O)NR₄, OC(O)NH, NR₄C(O)O, OC(O)NR₄, and NHC(O)O with     the proviso that L₂ does not contain a C(O)NH, C(O)NR₄, C(O)O, or     C(O)S unit; the 5-membered heteroaryls are selected from the group     consisting of imidazole, furane, thiophene, thiazole, isothiazole,     oxazole, isoxazole, 1,2,3-oxadiazole, 1,3,4-oxadiazole,     1,2,4-oxadiazole, 1,2,3-triazole, 1,3,4-triazole, 1,2,3-thiazole,     1,3,4-thiazole, and 1,2,4-thiazole; the 6-membered heteroaryls are     selected from the group consisting of pirydine, pirymidyne,     pirydazine, 1,2,4-triazine, and 1,3,5-triazine; and R₄ is selected     from the group consisting of H and C₁₋₄ alkyl.

In some embodiments, the compounds of formula (I) are used in various salt forms, including but not limited to the hydrochloride, hydrobromide, methanosulfonate, isethionate, tosylate, benzenesulfonate, lactate, citrate, formate, and acetate salts.

In various embodiments, the compounds of formula (I) have the following structures:

In other embodiments, compounds for use as described herein include compounds having the following structures:

In one embodiment, compounds for use as described herein include the compounds having the formula:

wherein R is selected from the group consisting of H, OH, OMe, and

In another embodiment, compounds for use as described herein include the compounds having the formula:

wherein X₁ is selected from the group consisting of H, Me, OMe, and Cl; and X₂ is selected from the group consisting of H, Me, and OMe.

In another embodiment, compounds for use as described herein include the compounds having the formula:

wherein X₁ is selected from the group consisting of H, Me, OMe, Cl, and CF₃; and X₂ is selected from the group consisting of H and Me.

In other embodiments, compounds for use as described herein include the compounds set forth below as described in the following U.S. patents:

U.S. Pat. No. 4,933,347

The above-named patent describes compounds having the following formula:

wherein each R₁ is H or two R₁ groups on the same amidine group together represent —(CH₂)_(m)—, wherein m=2, 3, or 4; R₂ is H, OCH₃, NO₂ or NH₂; R₃ is H, CH₃, or CH₂CH₃; n=2, 3, 4 or 5; and X is O, N or S; provided that when both R₁ and R₂ are H and X is O, then n cannot equal 5.

Particularly preferred are those compounds that have the para-amidine structure, as shown below:

wherein R₁, R₂, R₃, X, m and n have the same meanings as above.

Other compounds described have the formula:

wherein each R₁ is H or two R₁ groups on the same amidine group together represent —(CH₂)_(m)—, wherein m=2, 3 or 4; R₂ is H OCH₃, NO₂ or NH₂; R₃ is H, CH₃, or CH₂CH₃; n=2, 3, 4 or 5; and X is O, N or S; with the provisos that when both R₁ and R₂ are H, then X is N or S, and when R₂ is H and X is O, then two R₁ groups together represent —(CH₂)_(m)—, and n=3 or 4.

Particularly preferred of these compounds are those that have the para-amidine structure, as shown below:

wherein R₁, R₂, R₃, X, m and n and have the same meanings as above. Additionally, new compounds wherein n=6 are contemplated.

Methods for synthesizing the compounds above are described in U.S. Pat. Nos. 4,933,347. 4,933,347 is hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,206,236

The above-named patent describes compounds having the structure:

wherein X is O, N or S; R₁ is H or two R₁ groups on the same amidine group together represent —(CH₂)_(m)—, wherein m=2, 3 or 4; R₂ is H, NH₂, OCH₃, Cl, or NO₂; R₃ is H, CH₃ or CH₂ CH₃ and n=2-6, or pharmaceutically acceptable salts thereof, or more preferably a compound of formula:

wherein X, R₁, R₂, R₃, m and n have the foregoing meanings, or a pharmaceutically acceptable salt thereof.

U.S. Pat. No. 5,206,236 is hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,578,631

The above-named patent describes compounds having the formula:

wherein R₁ and R₂ are each independently selected from the group consisting of H or lower alkyl, or R₁ and R₂ together represent —(CH₂)_(m)— wherein m is from two to four; R₃ is H or lower alkyl; and X is C₁₋₁₂ linear or branched, saturated or unsaturated alkyl containing up to four double bonds (e.g., —(CH₂)_(n) wherein n is from 1-8, which is unsubstituted or substituted from 1 to 2 times with loweralkyl, and which is saturated or unsaturated and contains up to two double bonds); or a pharmaceutically acceptable salt thereof. Currently preferred are bis[5-(2-imidazolyl-2-benzimidazolyl]methane and 1,4-bis[5-(2-imidazolyl)-2-benzimidazolyl]butane, or pharmaceutically acceptable salts thereof.

Also described are compounds having the above formula wherein R₁ and R₂ together represent —(CH₂)_(m)— wherein m is from two to four; R₃ is H or loweralkyl; and X is selected from the group consisting of —CH₂—CH₂—CH₂—CH₂—, —CH—CH—CH₂—CH₂—, —CH₂—CH—CH—CH₂—, —CH—CH—CH—CH—, and any of the foregoing substituted from 1 to 2 times with loweralkyl; and the pharmaceutically acceptable salts thereof.

U.S. Pat. No. 5,578,631 is hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,602,172

The above-named patent describes compounds having the formula:

wherein R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; R₃ and R₄ are each independently selected from the group consisting of H, lower alkyl, oxyalkyl, alkylaryl, aryl, oxyaryl, aminoalkyl, aminoaryl, or halogen; and X and Y are located in the para or meta positions and are selected from the group consisting of H, loweralkyl, oxyalkyl, and

wherein each R₅ is independently selected from the group consisting of H, lower alkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂-C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₆ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl.

In some embodiments, two R₅ groups together represent

wherein m is from 1-3 and R₇ is H or —CONHR₈NR₉R₁₀, wherein R₈ is lower alkyl, and R₉ and R₁₀ are each independently selected from the group consisting of H and lower alkyl, although these compounds are not currently preferred.

In some embodiments, the compounds described has the structure formula above and include compounds wherein X and Y are located in the para position and are each

and wherein:

-   -   (a) R₁ is H, R₂ is H or loweralkyl, R₃ is H, R₄ is H, R₅ is H,         and R₆ is isoalkyl, such as isopropyl, isobutyl, isopentyl, and         the like;     -   (b) R₁ is H, R₂ is H, R₃ is H, R₄ is H, R₅ is H, and R₆ is C₃-C₈         alkoxyalkyl;     -   (c) R₁ is H, R₂ is H or loweralkyl, R₃ is H, R₄ is H, R₅ is H,         and R₆ is alkylhydroxy, such as ethylhydroxy, propylhydroxy,         butylhydroxy, pentylhydroxy, and hexylhydroxy;     -   (d) R₁ is H, R₂ is H or loweralkyl, R₃ is H, R₄ is H, R₅ is H,         and R₆ is propoxyethyl;     -   (e) R₁ is H, R₂ is H or loweralkyl, R₃ is H, R₄ is H, R₅ is H,         and R₆ is propoxyisopropyl;     -   (f) R₁ is H, R₂ is H or loweralkyl, R₃ is H, R₄ is H, R₅ is H,         and R₆ is aryl or alkylaryl; and     -   (g) R₁ is H, R₂ is H or loweralkyl, R₃ is H, R₄ is H, R₅ is H,         and R₆ is alkylcycloalkyl; and phamaceutically acceptable salts         thereof.

Methods of synthesizing the compounds described above are disclosed in U.S. Pat. Nos. 5,602,172. 5,602,172 is hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,668,167

The above-named patent describes compounds having the following formula:

wherein X is located in the para or meta positions and is loweralkyl, loweralkoxy, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, alkylaryl, halogen, or

wherein each R₂ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₂ groups together represent C₂-C₁₀ alkylene, or two R₂ groups together represent

wherein m is from 1-3 and R₄ is H,

or —CONHR₅NR₆R₇, wherein R₅ is loweralkyl, R₆ and R₇ are each independently selected from the group consisting of H and lower alkyl; each R₈ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl, or two R₈ groups together represent C₂-C₁₀ alkylene; R₉ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl; R₃ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl; R₁ is H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, alkylaryl, or halogen; or a pharmaceutically acceptable salt thereof.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 5,668,167, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,686,456

The above-named patent describes compounds have the formula:

wherein X and Y are located in the para or meta positions and are selected from the group consisting of H, loweralkyl, loweralkoxy, and

wherein each R₁ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₁ groups together represent C₂-C₁₀ alkyl, hydroxyalkyl, or alkylene, or two R₁ groups together represent

wherein m is from 1-3 and R₇ is H or —CONHR₈NR₉R₁₀, wherein R₈ is loweralkyl, and R₉ and R₁₀ are each independently selected from the group consisting of H and lower alkyl; R₂ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl; n is a number from 0 to 2 (where n is 0, the bond is direct covalent linkage between the rings); R₃ and R₄ are each independently selected from the group consisting of H, loweralkyl, loweralkoxy, alkylaryl, aryl, oxyaryl, aminoalkyl, aminoaryl, or halogen; and R₅ and R₆ are each independently selected from the group consisting of H, loweralkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; or a phamaceutically acceptable salt thereof.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 5,686,456, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,723,495

The above-named patent describes patents have the formula:

wherein R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl or alkylaminoalkyl;and X is C₁₋₁₂ linear or branched, saturated or unsaturated alkyl containing up to four double bonds; and Y is H or loweralkyl; or pharmaceutically acceptable salts thereof.

In another embodiment, the above-named patent describes compounds having the formula:

wherein R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl or alkylaminoalkyl; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; R₄ is —OH, or R₁ and R₄ together represent

Y is H or loweralkyl; n is an integer from 0 to 2; and A is a heterocyclic aromatic group selected from the group consisting of:

wherein R₆, R₇ and R₈ are each independently selected from the group consisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; R₉ is hydrogen, loweralkyl, hydroxy, aminoalkyl or alkylaminoalkyl; or pharmaceutically acceptable salts thereof.

In another embodiment, the above-named patent describes compounds having the formula:

wherein R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl or alkylaminoalkyl; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; R₄ is —OH, or R₁ and R₄ together represent

Y is H or loweralkyl; n is an integer from 0 to 2; or pharmaceutically acceptable salts thereof.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 5,723,495, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,127,554

The above-named patent describes compounds having the formula:

wherein R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; R₃ and R₄ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkylaryl, aryl, oxyaryl, aminoalkyl, aminoaryl, or halogen; and X and Y are located in the para or meta positions and are each selected from the group consisting of H, loweralkyl, oxyalkyl, and

wherein each R₅ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂ to C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₆ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; or a pharmaceutically acceptable salt thereof.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 6,127,554, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,172,104

The above-named patent describes compounds having the formula:

wherein R₁, R₂, R₃, and R₄ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, oxyaryl, oxyarylalkyl, or halogen; A and B are each selected from the group consisting of H, loweralkyl, oxyalkyl, and

wherein each R₅ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂ to C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₆ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; or a pharmaceutically acceptable salt thereof.

In another embodiment, this patent describes compounds having the formula:

wherein R₁, R₂, R₃, and R₄ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, oxyaryl, oxyarylalkyl, or halogen; A and B are each selected from the group consisting of H, loweralkyl, oxyalkyl, and

wherein each R₅ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂ to C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₆ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; or a pharmaceutically acceptable salt thereof.

In another embodiment, this patent describes compounds having the formula:

wherein R₁, R₂, R₃, and R₄ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, oxyaryl, oxyarylalkyl, or halogen; A and B are each selected from the group consisting of H, loweralkyl, oxyalkyl, and

wherein: each R₅ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂ to C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₆ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; or a pharmaceutically acceptable salt thereof.

In another embodiment, the above-named patent describes a compound having the formula:

wherein R₁, R₂, R₃, and R₄ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, oxyaryl, oxyarylalkyl, or halogen; A and B are each selected from the group consisting of H, loweralkyl, oxyalkyl, and

wherein: each R₅ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂ to C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₆ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; or a pharmaceutically acceptable salt thereof.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 6,172,104, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,326,395

The above-entitled patent describes compounds having the formula:

wherein R₁ and R₂ may be the same or different and selected from the group consisting of H, loweralkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, halogen, oxyalkyl, oxyaryl, and oxyarylalkyl; and wherein Y₅ and Y₆ are present in the meta or para positions and may the same or different and are represented by the formula (a) or (b) selected from the group consisting of:

wherein each R₂₂ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₂₂ groups together represent C₂-C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₂₃ is H, hydroxy, loweralkyl, alkoxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; and

wherein Y₃ is selected from the group consisting of NR′″ and O; wherein R′″ is selected from the group consisting of H and loweralkyl; and wherein Y₄ is represented by the formula:

wherein R₂₀ is selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl; wherein R₂₁ is selected from the group consisting of hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, and alkylaryl.

In another embodiment, the above-named patent discloses compounds having the formula:

wherein Y₁₅ and Y₁₆ may be the same or different and represented by the formula:

wherein: each R₂₂ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkylamino, cycloalkyl, aryl, or alkylaryl or two R₂₂ groups together represent C₂-C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₂₃ is H, hydroxy, loweralkyl, alkoxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl.

In another embodiment, the above-named patent describes compounds having the formula:

wherein each R₂₅ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₂₅ groups together represent substituted or unsubstituted C₂-C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₂₆ is H, hydroxy, loweralkyl, alkoxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; R″ is hydroxy, alkoxyalkyl, hydroxyalkyl, alkoxyaryl, aryl, or the substituent selected from the formula (i) and (ii) consisting of:

wherein n and m may be independently selected and each range from 0 to 6; each R₂₂ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₂₂ groups together represent C₂-C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₂₃ is H, hydroxy, loweralkyl, alkoxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl.

In another embodiment, the above-named patent describes compounds having the formula:

wherein n is from 2 to 6; X is selected from the group consisting of O, NH, and S; Y₉ and Y₁₀ may be in the meta or para position, are independently selected and are each represented by the formula:

wherein each R₃₀ is selected from the group consisting of H, hydroxy, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; and wherein each of the two R₃₀ groups together may represent C₂-C₁₀ alkyl, hydroxyalkyl, or alkylene; wherein R₃₁ is selected from the group consisting of H, hydroxy, loweralkyl, alkoxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; wherein R₃ and R₄ may be the same or different and are selected from the group consisting of H, amino nitro, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl.

In another embodiment, the above-named patent describes compounds having the formula:

wherein X may be O, NH, or S; n and m may be the same or different and range from 2 to 6; wherein Y₁₁ and Y₁₂ may be the same or different and represented by the formula:

wherein each R₃₀ is selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; and wherein each of the two R₃₀ groups together may represent represent C₂-C₁₀ alkyl, hydroxyalkyl, or alkylene; wherein R₃₁ is selected from the group consisting of H, hydroxy, loweralkyl, alkoxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; wherein R₅ is selected from the group consisting of H, hydroxy, and

wherein n ranges from 0 to 3.

In another embodiment, the above-named patent describes compounds having the formula:

wherein X is C₁ to C₁₂ linear or branched, saturated or unsaturated alkyl containing up to four double bonds, or is substituted or unsubstituted aryl; wherein Y₁₃ and Y₁₄ may be the same or different and are represented by the formula:

wherein R₄₀ and R₄₂ are each independently selected from the group consisting of H, loweralkyl, cycloalkyl, substituted aryl, and unsubstituted aryl, or wherein R₄₀ and R₄₂ together may represent C₂-C₁₀ alkyl, hydroxyalkyl, alkylene, substituted aryl, or unsubstituted aryl; and wherein R₄₁ may be H, hydroxy, loweralkyl, alkoxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 6,326,395, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,423,737 B2

The above-named patent describes compounds having the formula:

wherein R₂ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl or alkylaminoalkyl; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; n is from 2 to 6; X is O or S; and Y is H or loweralkyl; or pharmaceutically acceptable salts thereof.

In another embodiment the above-named patent describes compounds having the formula:

wherein R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl or alkylaminoalkyl; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; R₄ is —OY, or R₁ and R₄ together represent

Y is H or loweralkyl; n is an integer from 0 to 2; and A is a heterocyclic aromatic group selected from the group consisting of:

wherein R₆ and R₇ are each independently selected from the group consisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; or pharmaceutically acceptable salts thereof.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 6,423,737, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,649,652

The above-named patent describes compounds having the formula:

wherein X may be O, S, or NR′ wherein R′ is H or loweralkyl; R₁ and R₂ may be independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; R₃ and R₄ are each independently selected from the group consisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, and oxyarylalkyl; R₅ is represented by a formula selected from the group consisting of:

wherein: X₁, X₂, and X₃ are independently selected from O and S; and R₆ and R₇ are independently selected from the group consisting of loweralkyl, aryl, alkylaryl, oxyaryl, an ester-containing substituent, and oxyalkyl; or a pharmaceutically acceptable salt thereof. Preferably, R₆ and R₇ are independently selected from the group consisting of CH₃, CH₂CCl₃, CH₂CH₃,

In one preferred embodiment, each of the substituents present on the compound represented by the formula:

are present on the para positions of the aromatic groups, although these substituents may be present in the meta positions.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 6,649,652, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,635,668

The above-named patent describes compounds having the formula:

wherein A and B are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy, carboxy, and compounds of formula:

subject to the proviso that at least one of A and B is such compound; R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl, hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on the same amidine group together represent —(CH₂)_(m)— wherein m is 2, 3, or 4; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; n is from 2 to 6; and X is O, NH, or S; or a pharmaceutically acceptable salt thereof. In one embodiment, R₁, R₂ and R₃ are H; X is O; and n is 5.

In another embodiment, the above-named patent describes compounds having the formula:

wherein: A and B are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy, carboxy, and compounds of formula:

subject to the proviso that at least one of A and B is such compound; R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl, hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on the same amidine group together represent —(CH₂)_(m)— wherein m is 2, 3, or 4; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; X is linear or branched, saturated or unsaturated C1-C12 alkyl containing up to 4 double bonds; or X is a heterocyclic aromatic group selected from the group consisting of:

wherein R₆, R₇, and R₈ are each independently selected from the group consisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; R₉ is hydrogen, loweralkyl, hydroxy, aminoalkyl or alkylaminoalkyl; or the pharmaceutically acceptable salts thereof.

In another embodiment, the above-named patent describes compounds having the formula:

wherein: A and B are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy, carboxy, and substituents of formula:

subject to the proviso that at least one of A and B is such substituent; R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl, hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ groups on the same amidine group together represent —(CH₂)_(m)— wherein m is 2, 3, or 4; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; or two R₁ groups on the same amidine group together represent

n is an integer from 0 to 2; and A is a heterocyclic aromatic group selected from the group consisting of:

wherein R₆, R₇, and R₈ are each independently selected from the group consisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; R₉ is hydrogen, loweralkyl, hydroxy, aminoalkyl or alkylaminoalkyl; and the pharmaceutically acceptable salts thereof.

In another embodiment, the above-named patent describes compounds having the formula:

wherein A and B are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy, carboxy, and substituents of formula:

subject to the proviso that at least one of A and B is such a substituent; R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl, hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on the same amidine group together represent —(CH₂)_(m)— wherein m is 2, 3, or 4; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; or two R₁ groups on the same amidine group together represent

and the pharmaceutically acceptable salts thereof.

In another embodiment, the above-named patent describes compounds having the formula:

wherein A and B are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy, carboxy, and substituents of formula:

subject to the proviso that at least one of A and B is such a substituent; R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, aryl, alkylaryl, aminoalkyl, aminoaryl, halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; R₃ and R₄ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkylaryl, aryl, oxyaryl, aminoalkyl, aminoaryl, or halogen; and each R₅ is independently selected from the group consisting of H, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂ to C₁₀ alkyl, hydroxyalkyl, or alkylene; and R₆ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; or a pharmaceutically acceptable salt thereof.

In another embodiment, the above-named patent describes compounds having the formula:

wherein A and B are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy, carboxy, and substituents of formula:

subject to the proviso that at least one of A and B is such a substituent; R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl, hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on the same amidine group together represent —(CH₂)_(m)— wherein m is 2, 3, or 4; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; or two R₁ groups on the same amidine group together represent

X is O, S or NH; n is an integer from 1 to 8; and the pharmaceutically acceptable salts thereof.

U.S. Pat. No. 5,668,167 is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,613,787

The above-named patent describes compounds having the formula:

wherein X is selected from the group consisting of O, S, and NH; Y is CH or N; A is CH or N; B is selected from the group consisting of NH, O or S; R₁ is selected from the group consisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, and oxyarylakyl; R₂ and R₉ are each independently selected from the group consisting of H, H₂, hydroxy, lower alkyl, cycloalkyl, aryl, alkylaryl, alkoxyalkyl, hydroxycycloalkyl, alkoxycycloalkoxy, hydroxyalkyl, aminoalkyl and alkylaminoalkyl; and R₃, R₄, R₁₃ and R₁₄ are each independently selected from the group consisting of H, lower alkyl, alkoxyalkyl, cycloalkyl, aryl, alkylaryl, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl, or R₃ and R₄ together or R₁₃ and R₁₄ together represent a C₂ to C₁₀ alkyl, hydroxyalkyl, or alkylene, or R₃ and R₄ together or R₁₃ and R₁₄ together are:

wherein n is a number from 1 to 3, and R₁₀ is H or —CONHR₁₁NR₁₅R₁₆, wherein R₁₁ is lower alkyl and R₁₅ and R₁₆ are each independently selected from the group consisting of H and lower alkyl; L is selected from the group consisting of:

wherein R₅, R₆, R₇, and R₈ are each individually selected from the group consisting of H, alkyl, halo, aryl, arylalkyl, aminoalkyl, aminoaryl, oxoalkyl, oxoaryl, and oxoarylalkyl; and wherein the compound of Formula I binds mixed-sequence DNA in the minor groove in a dimer formation.

In a preferred embodiment the compound described above is a dication, L is:

A is N; B is NH; X is O; Y is CH; R₁, R₂, R₄, R₅, R₆, R₇, R₈, R₉ and R₁₄ are each H; and R₃ and R₁₃ are each H₂.

Methods of synthesizing the compounds described above are described in U.S. Pat. No. 6,613,787, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 4,619,942

The above named patent describes the following compounds:

1 Benzamidine

2 Pentamidine

3 1-(4-Amidinophenoxy-6-phenoxyhexane

4 1-(4-Amidinophenoxy)-8-phenoxyoctane

5 1,4-Di(4-amidinophenoxy)-2-butanol

6 a,a′-Bis(4-amidino-2-iodophenoxy)-m-xylene

7 Bis(5-amidino-2-benzimidazolyl)methane

8 1,2-Bis(5-amidino-2-benzimidazolyl)ethane

9 5-Amidinoindole

10 5-Amidinobenzofuran

11 5-Amidinobenzimidazole

12 5-Amidino-1-methylindole

13 5-Amidino-1-(4-amidinobenzyl)indole

U.S. Pat. No. 4,619,942 is hereby incorporated by reference in its entirety.

U.S. Pat. No. 4,324,794

The above-named patent describes compounds having the formula:

wherein R=H or C(NH)NH₂ and X=alkane-1, ω-diyl, or 2-hydroxybutane-1,4-diyl.

In another embodiment, the above-named patent describes compounds having the formula:

wherein n=1 or 2.

In another embodiment, the above-named patent describes compounds having the formula:

wherein X=CH or N; X¹=O, NH, NMe, or NCH₂C₆H₄C(NH)NH₂₋₄).

Methods for synthesizing the compounds disclosed above are described in U.S. Pat. No. 4,324,794, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,699,862

The above-named patent describes compounds having the formula:

wherein R¹=H or alkyl; R³, R⁴, R⁵, R⁸, R⁹, and R¹⁰=H or alkyl; alternatively, R³R⁴ and R⁸R⁹ form an imidazolinyl group; R¹⁴=H, NHCO(CH₂)_(m)R²⁰, (CH₂)_(m)R²⁰, CHMeR²⁰, (CH₂)_(m)(C₆H₃)R¹⁷, (CH₂)_(m)(C₆H₃)R²⁰, (CH₂)_(m)(heterocyclyl)R¹⁷, (CH₂)_(m)(heterocyclyl)R²⁰, CH₂CH:CHR²⁰, (CH₂)_(m)CONHCHR²⁰R²¹, (CH₂)_(m)CONHCH₂CONHCHR²⁰R²¹; R¹⁷=H, halo, alkyl, CF₃, CN, NO₂, N(R¹)₂, OH, alkylalkoxy; R²⁰=CO₂R¹, CH(OH)CH₂OH, CONH₂, CHO; R²¹=H, alkyl, (CH₂)_(n)R²², CHMeCH₂CO₂R¹, CH₂Ph; R²²=H, NH₂, OR¹, SR¹, CN OCH₂Ph, O(CH₂)_(m)OR¹, CO₂R¹, thienyl, tetrahydropyranyl, CH(OH)CH₂OH, COCMe:CH₂, NHCO₂CH₂Ph, SO₂R¹; m=1-3; and n=1-5.

Methods for synthesizing the compounds disclosed above are described in U.S. Pat. No. 6,699,862, which is hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,248,673

The above-named patent discloses the compound having the formula:

U.S. Pat. No. 5,248,673 is hereby incorporated by reference in its entirety.

U.S. Pat. No. 6,106,866

The above-named patent describes the compound of formula:

U.S. Pat. No. 6,106,866 is hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,597,573

The above-named patent describes compounds having the formula:

wherein R¹-R⁶=(un)substituted alkyl, alkene, or alkyne and E=O or NH. U.S. Pat. No. 5,597,573 is hereby incorporated by reference in its entirety.

Other U.S. patents also disclose compounds useful in the present invention, some of which are addressed above. The patents include U.S. Pat. Nos. 6,503,940; 6,486,200; 6,025,398; 6,008,247; 5,843,980; 6,214,883; 5,668,166; 5,627,184; 5,622,955; 5,606,058; 5,521,189; 5,428,051; 5,202,320; 4,963,589; 4,397,863; 4,324,794; 6,204,279; and 6,245,746, all of which are hereby incorporated by reference in their entirety.

In other embodiments, compounds for use as described herein include the compounds set forth below as described in the following published international PCT applications, published foreign patent applications, and published articles:

PCT International Application No. WO 2004/006849 A2

The above-named PCT application describes compounds having the following formulae:

PCT Application No. WO 2004/006849 is hereby incorporated by reference in its entirety.

“Antileishmanial Activities of Several Classes of Aromatic Dications,” Brendle, James J., et al., Antimicrobial Agents and Chemotherapy (2002), 46(3), 797-807.

The above-named article describes compounds having the formulae:

This article is hereby incorporated by reference in its entirety.

PCT International Application No. WO 2001/021585 A2

The above-named PCT application describes the compound having the formula:

This application is hereby incorporated by reference in its entirety.

PCT International Patent Application WO 2000/010990 A2

The above-named application discloses compounds having the formula:

wherein each R independently is H, alkyl, oalkoxy, C(NR⁵)NR⁵R⁶; R¹-R⁴=H, halo, alkyl, alkoxy, etc.; R⁵=H, alkyl, alkoxy, aryl, etc.; R⁵=alkylene, etc.; R⁶=H, OH, alkyl, alkoxy, etc.; and Z=O or S.

In one embodiment, the compound has the formula:

PCT Application No. WO 2000/010990 A2 is hereby incorporated by reference in its entirety.

“Reactions of 1,2,5-thiadiazole-3,4-dicarbonitrile,” Moerkved, Eva H., et al., Acta Chemica Scandinavica (1994), 48(4), 372-6

The above-named article discloses compounds having the formulas:

This article is hereby incorporated by reference in its entirety.

“Model Studies On The Structure ofP(amide oximes) and Their cyclodehydration Reactions Leading to Poly(1 2,4-oxadiazoles),” Jung, Jin Chul, et al., Journal of Polymer Science, Part A: Polymer Chemistry (1993), 31(13), 3351-9

The above-named article discloses compounds having the following structures:

This article is hereby incorporated by reference in its entirety.

“Synthesis of Mixed 1,2,4-oxadiazoles by Reaction of Perfluorinated Nitrites with benz- and Terephthalamidoximes,” Kabakchi, E. V., et al., Izvestiya Akademi Nauk, Seriya Khimicheskaya (1992), (8), 1863-70

The above-named article discloses compounds having the following formulae:

This article is hereby incorporated by reference in its entirety.

“Growth Inhibition and Induction of Cellular Differentiation of Human Myeloid Leukemia Cells in Culture by Carbamoyl Congeners of Ribavirin,” Sanghvi, Yogesh S., et al., Journal of Medicinal Chemistry (1990), 33(1), 336-44.

The above-named article discloses compounds having the following formula:

wherein either X=X1=N; X=N and X1=CH; X=X1=CH; or X=CNH2 and X1=N. In some embodiments, the compounds have one of the following formulae:

The above-named article is hereby incorporated by reference in its entirety.

French Patent No. 2601010 A1

The above-named French patent discloses compounds having the following formula:

wherein R¹ and R² are separately selected from the group consisting of H, (un)substituted alkyl, cycloalkyl, aryl, aralkyl, alkoxy, alkanoyl, and aroyl; or NR¹R² may be an (un)substituted heterocyclyl; R³ is selected from the group consisting of (un)substituted alkyl, cycloalkyl, NH₂; X=SO, NOR⁴; R⁴=H, alkyl, cycloalkyl, etc.; Z₁-Z⁴=CH, CR, N; R=H, halo, alkyl, alkoxy, etc.

The above-named patent also describes compounds having the formula:

wherein R⁶ is selected from the group consisting of cyano, C(S)NH₂, and C(SO)NH₂.

In some embodiments, the above-named patent describes compounds having the formula:

Methods for synthesizing the compounds disclosed above are described in French Patent No. 2601010 A1, which is hereby incorporated by reference in its entirety.

“Direct Synthesis of Pyrrole Nucleosides by The Stereospecific Sodium Salt Glycosylation Procedure,” Ramasamy Kandasamy, Journal of Heterocyclic Chemistry (1987), 24(3), 863-8.

The above-named article describes compounds have the formula:

wherein R is H or cyano.

In another embodiment, the above-named article describes compounds having the formula:

wherein R¹=H and Z=O, S, or NOH; or R¹=CONH₂ and Z=O; or R¹=CSNH₂ and Z=S; or R¹=C(NOH)NH₂ and Z=NOH.

In some embodiments, the above-named article discloses compounds having the structure:

Methods for synthesizing the compounds disclosed above are described in the above-named article, which is hereby incorporated by reference in its entirety.

“Synthesis of Pyridyloxadiazoles, 2,2-(Oxadiazolyl)Pyridines and 2,6-bis(Oxadiazolyl)Pyridines As Analogs of Pyridinolcarbamate,” Suarez, Cecilia, et al. Journal of Heterocyclic Chemistry (1978), 15(7), 1093-6.

The above-named article discloses compounds having the following formulae:

The above-named article is hereby incorporated by reference in its entirety.

“Hydroxylamine Derivatives As Potential Antimalarial Agents. 3. 1,2,4-Oxadiazoles,” Hynes, John B., et al. Journal of Medicinal Chemistry (1972), 15(11), 1198-1200.

The above-named article discloses the compound having the formula:

The above-named article is hereby incorporated by reference in its entirety.

“Picrylamino-substituted Heterocycles. II. Furazans,” Coburn, Michael D. Journal of Heterocyclic Chemistry (1968) 5(1), 83-7.

The above-named article discloses the compound having the formula:

The above-named article is hereby incorporated by reference in its entirety.

“Structure-Activity Relationships of Analogs of Pentamidine Against Plasmodium falciparum and Leishmania mexicana amazonensis,” Bell, Constance A., et al., Antimicrobial Agents and Chemotherapy, July 1990, pp. 1381-1386.

The above-named article discloses compounds having the formula:

wherein n is from 2 to 6.

In another embodiment, the above-named article discloses compounds having the formula:

wherein n is from 3 to 6.

In another embodiment, the above-named article discloses compounds having the formula:

wherein n is from 2 to 5 and X may be NO₂ when n is 2, 4, or 5; X may be NH₂ when n is 2, 3, or 4; X may be OCH₃ when n is 3, 4, or 5; X may be Cl when n is 4 or 5; and X may be Br when n is 5.

In another embodiment, the above-named article discloses compounds having the formula:

wherein n is from 3 to 6.

In another embodiment, the above-named article discloses compounds having the formula:

wherein n is from 2 to 6 and when n is 2, 4, 5 or 6, X is NH₂ and when n is 3 or 5, X I NO₂.

In another embodiment, the above-named article discloses compounds having the formula:

wherein n is from 3 to 5, X is H or OCH₃, and Y is H.

The above-named article is hereby incorporated by reference in its entirety.

PCT International Application No. WO 01/30757

The above-named PCT application discloses efflux pump inhibitors having the formula:

wherein R¹ and R² independently represent each hydrogen, halogeno, carboxy, etc.; J¹ represents 5- or 6-membered heteroaryl; W¹ represents —CH═CH—, —CH═CH—, —CH₂CH₂—, etc.; A¹ represents phenylene, pyridinedyl, furandyl, etc.; G¹ represents oxygen, carbonyl, ethynyl, etc.; p is an integer of from 0 to 3; G² represents phenylene, flirandyl, tetrahydrofurandyl, etc.; G³ represents —CH₂— or a single bond; m and n represent each an integer of 0 or 1; and Q¹ represents an acidic group. PCT Application No. WO 01/30757 is hereby incorporated by reference in its entirety.

PCT International Patent Application No. WO 02/087589

The above-named PCT application describes efflux pump inhibitors having the formula:

wherein R¹ and R² each represent hydrogen, a halogen atom, a hydroxyl group or the like; W¹ represents —CH═CH—, —CH₂O—, —CH₂CH₂— or the like; R³ represents hydrogen, a halogen atom, a hydroxyl group or an amino group; R⁴ represents hydrogen, a group of —OZ₀₋₄R⁵(where Z₀₋₄ represents an alkylene group or a fluorine-substituted alkylene group or a single bond and R⁵ represents a cyclic alkyl group, an aryl group or the like) or the like; W² represents a single bond or —C(R⁸)═C(R⁹)-(where R⁸ and R⁹ each represent hydrogen, a halogen atom, a lower alkyl group or the like) and Q represents an acidic group, with the proviso that W² and Q may together form a heterocyclic ring of vinylidene thiazolidinedione or an equivalent thereof; m and n each represent an integer of 0 to 2 and q represents an integer of 0 to 3.

PCT Application No. WO 02/087589 is hereby incorporated by reference in its entirety.

“Synthesis and anti-Pneumocystis carinii Activity of Conformationally Restricted Analogues of Pentamidine.” Tao, Bin et al., European Journal of Medicinal Chemistry (1999), 34(6), 531-538.

The above-named article discloses compounds having the following structures:

The article is hereby incorporated by reference in its entirety.

“Structure-in Vitro Activity Relationships of Pentamidine Analogs and Dication-substituted bis-benzimidazoles As New Antifungal Agents,” Del Poeta, Maurizio, et al., antimicrobioal Agents and Chemotherapy (1998), 42(10), 2495-2502.

The above-named article discloses compounds having the following structures:

The article is hereby incorporated by reference in its entirety.

“Synthesis, Characterization, and Structure-activity Relationships of Amidine-substituted (bis) Benzylidene-cycloketone Olefin Isomers As Potent and Selective Factor Xa Inhibitors,” Guilford, William J., et al., Journal of Medicinal Chemistry (1999), 42(26), 5415-5425.

The above-named article discloses compounds having the following structures:

The article is hereby incorporated by reference in its entirety.

“Derivatives of 5-amidine Indole As Inhibitors of Thrombin Catalytic Activity,” Iwanowicz, Edwin J., et al., Bioorganic & Medicinal Chemistry Letters (1996), 6(12), 1339-1344.

The above-named article discloses compounds having the structure:

The article is hereby incorporated by reference in its entirety.

“On The Structure-activity Relationship of Histamine H2-receptor Antagonists Based On The X-ray Crystal Structures and 1H-NMR Spectra of Amidine Derivatives.” Ishida. Toshimasa, et al., Molecular Pharmacology (1987), 31(4), 410-16.

The above-named article discloses compounds having the structure:

The article is hereby incorporated by reference in its entirety.

“Structure-activity Relationships in Distamycin A Analogs: Effect of Alkyl Groups On The Pyrrole Nitrogen At The Non-amidine End of The Molecule Combined with Methyl Elimination in The Following Ring.” Grehn, Leif, et al., Acta Chemica Scandinavica, Series B: Organic Chemistry and Biochemistry (1986), B40(2), 145-51.

The above-named article discloses compounds having the structure:

The article is hereby incorporated by reference in its entirety.

“Inhibitory Activity of Diarylamidine Derivatives On Murine Leukemia L1210 Cell Growth,” Balzarini, Jan, et al., Investigational New Drugs (1983), 1(2), 103-15.

The above-named article describes compounds having the following formula:

wherein X=NH, O S, SO₂, or CH₂; Y=CH, CNH₂, N, etc.; R¹ and R² separately are amidino, imidazolino, etc.; Z=CH:CH, PhO, CONH, NH, etc; and n=0 or 1.

In another embodiment, the above-named article describes compounds having

the formula: wherein R¹ and R²=amidino or imidazolino and Z=CH:CH, NHN:N, etc.

In another embodiment, the above-named article describes compounds having the formula:

wherein X=O, S or NH; Y=CH, CMe, or N; and R¹ and R²=amidino or imidazolino.

In another embodiment, the above-named article describes compounds having the formula:

wherein X=NH; Y=CH; Z=CH:CH; R¹ and R²=imidazolino; and n=0 or 1.

The above-named article is hereby incorporated by reference in its entirety.

“Structure-activity Relationships of Pyrrole Amidine Antiviral Antibiotics. III: Preparation of Distamycin and Congocidine Derivatives Based On 2,5-disubstituted Pyrroles.” Bialer, Meir, et al., Journal of Pharmaceutical Sciences (1980), 69(11), 1334-8.

The above-named article discloses compounds having the formula:

wherein R=NO₂ or HCONH.

In another embodiment, the above-named article discloses compounds having the formula:

wherein R¹=NO₂, H₂NC(:NH)NHCH₂CONH.

“Structure-activity Relationships of Pyrrole Amidine Antiviral Antibiotics. 2. Preparation of Mono- and Tripyrrole Derivatives of Congocidine,” Bialer, Meir, et al., Journal of Medicinal Chemistry (1980), 23(10), 1144-8.

The above-named article discloses the compound having the following formula:

In another embodiment, the above-named article discloses compounds having the formula:

wherein R is C(:NH)NH₂ or CN and X is CH₂, CH₂CH₂, or CHMeCH₂.

Methods for synthesizing the compounds disclosed above are described in the above-named article, which is hereby incorporated by reference in its entirety.

“Synthesis of Bis-substituted Amidinobenzothiazoles As Potential Anti-HIV Agents.” Racane, Livio, et al., Heterocycles (2001), 55(11), 2085-2098.

The above-named article describes compounds have the formula:

wherein X=O, S; R=Me₂CH; R¹=H or RR¹=CH₂CH₂.

In another embodiment, the above-named article describes compounds having the formula:

wherein X=O, S; R=Me₂CH; R¹=H or RR¹=CH₂CH₂.

In another embodiment, the above-named article describes compounds having the formula:

wherein X=O or S.

In another embodiment, the above-named article describes compounds having the formula:

wherein X=O or S.

In another embodiment, the above-named article describes compounds having the formula:

wherein X=O or S.

Methods for synthesizing the compounds disclosed above are described in the above-named article, which is hereby incorporated by reference in its entirety.

“Noncovalent Interactions Between Tetrazole and An N,N′-Diethyl-Substituted Benzamidine,” Peters, Lars, et al., Journal of Organic Chemistry I(2001), 66(10), 3291-3298.

The above-name article discloses compounds having the following formulas:

The article is hereby incorporated by reference in its entirety.

PCT International Application No. WO 98/07420 A1

The above-named PCT application describes compounds having the formula:

wherein A=O, S, or NR; R=C₁₋₈ alkyl; D=O, S, or NR⁷; W=N, CH, or CR⁸; R¹ and R³ are independently H, (un)substituted, straight or branched, cyclic or acyclic satd., or unsatd. C₁₋₁₄ alkyl; R²=Q(X³)—NR⁵—W²—R⁶; W²=CO, SO₂, CONH, S(O), or single bond; Q=(un)substituted (CH₂)_(z), (CH₂)_(m)-Q¹-(CH₂)₁, z=1-12; when z>1, one or more CH₂ groups may be replaced by O, S, or substituted N; 1 and m are independently 0-5; Q¹=C₃₋₁₂ (un)satd. carbocyclic or heterocyclic ring; X³=H, C₁₋₈ alkyl, aryl, C₁₋₈ alkoxy, OH, CF₃, etc; R⁴=NR⁹R¹⁰, or NR¹¹—C(:A¹)-NR⁹R¹⁰; A¹=O, S, NH, or R¹²; R¹²=H, C₁₋₈ alkyl, or aryl; R⁵-R⁹, R¹¹, and R¹² are independently any group R¹, aryl, or heteroaryl; R¹⁰=H, straight or branched, cyclic or acyclic, satd. or unsatd. C₁-C₁₂ alkyl, (un)substituted aryl, arloxyalkyl, 2- or 3-tetrahydrofurfuryl, (CH₂)₂₋₁₂—OH, amidoalkyl; NR⁹R¹⁰=3-10-membered ring; pure or partially separated stereoisomers or racemic mixtures thereof; and free bases or pharmaceutically acceptable derivatives thereof.

PCT Application No. WO 98/07420 A1 is hereby incorporated by reference in its entirety.

“Synthesis of Diphenyl Bisamidines As Potential Amebicides.” Venugopalan, B., European Journal of Medicinal Chemistry (1996), 31(6), 485-488.

The above-named article describes the compound of formula:

The article is hereby incorporated by reference in its entirety.

“Pentamidine Congeners. 2,2-Butene-Bridged Aromatic Diamidines and Diimidazolines As Potential Anti-Pneumocystis carinii Pneumonia Agents,” Donkor, Isaac O., et al., Journal of Medicinal Chemistry (1994), 37(26), 4554-7.

The above-named article describes compounds having the formula:

wherein R=C(:NH)NH₂ or 2-imidazolin-2-yl and R¹=H or OMe. The article is hereby incorporated by reference in its entirety.

“Synthesis of 1-aryl-2-arylamino-6-aryliminotetrahydro-1,3,5-triazine-4-thiones from 6aSiv-2,5-bisarylamino[1,2,4]Dithiazolo[2,3-b][1,2,4]Dithiazoles by Reaction with Amines,” Joshua, C. P., et al. Organic Chemistry Including Medicinal Chemistry (1993), 32B(8), 879-81.

The above-named article describes compounds having the following formulae:

The article is hereby incorporated by reference in its entirety.

“Pentamidine Congeners. 1. Synthesis of Cis- and Trans-butamidine Analogs As Anti-Pneumocystis carinii Pneumonia Agents,” Donkor, Isaac O., Bioorganic & Medicinal Chemistry Letters (1993), 3(6), 1137-40.

The above-named article describes compounds having the formula:

wherein R=NH₂. Also disclosed are compounds having the formula:

The article is hereby incorporated by reference in its entirety.

“Structure, DNA Minor Groove Binding, and Base Pair Specificity of Alkyl- and Aryl-linked Bis(Amidinobenzimidazoles) and Bis(Amidinoindoles),” Fairley, Terri A., Journal of Medicinal Chemistry (1993), 36(12), 1746-53.

The above-named article describes compounds having the formula:

wherein X=(CH₂)_(n) or phenylene and n=1-6.

In another embodiment, the above-named article describes compounds having the formula:

wherein n=3-6.

The above named article is hereby incorporated by reference in its entirety.

European Application No. 89-810491

The above-named European application describes compounds having the formula:

wherein one of X₁-X₃ and one of X₄-X₆ is CH and the others are CH or N; Y=O, S or NR⁵; Z=O, S, or NR⁶; R¹ and R³ are H, alkyl, aryl, etc.; and R² and R⁴-R⁶ are H or alkyl. Alternatively, NR¹R² and NR³R⁴ are heterocyclyls.

The above-named application is hereby incorporated by reference in its entirety.

Indian Patent Application No. 157285 A

The above-named Indian patent application describes compounds having the formula:

wherein X and X¹ are alkyl, alkoxy, halo, CF₃, SO₃H, SO₂Me, etc.; Y and Y¹ are halo, alkyl, etc.; R¹=H or (un)substituted alkyl; and R² and R³=H, (un)substituted alkyl, or substituted acyl. Alternatively, NR²R³ may form a heterocycle and R¹R² may form an (un)substituted N-containing heterocycle.

Indian Patent Application No. 157285 A is hereby incorporated by reference in it entirety.

Indian Patent Application No. 155439 A

The above-named Indian patent application describes compounds having the formula:

wherein R¹=H or alkyl; R² and R³=H or alkyl. Alternatively, R²R³N may form a heterocycle.

In another embodiment, the above-named application describes the compound having the formula:

Indian Patent Application No. 155439 A is hereby incorporated by reference in its entirety.

German Patent Application No. DE 3343815 A1

The above-named German patent application describes compounds having the formula:

wherein R¹=H or (un)substituted C₁₋₅ alkyl; R² and R³=H, (un)substituted C₁₋₅ alkyl, or C₁₋₅ alkanoyl; alternatively, R²R³N=heterocyclyl; alternatively, R¹CNR²=heterocyclyl; alternatively, R³=R² or C₂₋₅ carbalkoxy; R⁴ and R⁵=C₁₋₅ alkyl, alkoxy, halo, or SO₂Me; and R⁶ and R⁷=H or halo.

German Patent Application No. DE 3343815 A1 is hereby incorporated by reference in its entirety.

Indian Patent Application No.153442

The above-named Indian patent application describes compounds having the formula:

wherein R=H, halo, alkoxy, NH₂ mOnoalkylamino, dialkylamino, or N heterocyclyl optionally containing O, S, or N; R¹=H, alkyl, alkoxy, halo, NO₂, or NH₂; R²=H, (un)substituted alkyl; R³ and R⁴=H or alkyl; alternatively, C(R²)R³N=heterocyclyl; alternatively, NR³R⁴=heterocyclyl.

Methods for synthesizing the above compounds are described in Indian Patent Application No. 153442, which is hereby incorporated by reference in its entirety.

German Patent Application No. DE 3305329 A1

The above-named German application describes compounds having the formula:

wherein R=H; R²=OC(O); R¹=H, alkyl, alkoxy, halo, NO₂, or amino; R²=H or (un)substituted alkyl; R³ and R⁴=alkyl; alternatively, R³R⁴N=heterocyclyl; X=O, N, NH; X¹=O, H, halo, alkoxy, amino heterocyclyl; and dotted lines represent optional double bonds.

German Patent Application No. DE 3305329 A1 is hereby incorporated by reference in its entirety.

“Synthesis of Bisamidine Derivatives of Diphenylamine As Potential Anthelmintic Agents,” Shukla, J. S., et al., Indian Journal of Chemistry, Section B: Organic Chemistry Including Medicinal Chemistry (1981), 20B(12), 1072-4.

The above-named article describes the compound of formula:

The article is hereby incorporated by reference in its entirety.

“Effect of Aromatic Bisamidines On Blood Coagulation and Fibrinolysis.” Hauptmann. J., et al., Acta Biologica et Medica Germanica (1976), 35(5), 635-44.

The above-named article describes the compound of formula:

The article is hereby incorporated by reference in its entirety.

“Application of Molecular Topology to The Predction of Antifuingal Activity for a Set of Dication-substituted Carbazoles, Furans and Benzimidazoles,” Garcia-Domenech, et al. THEOCHEM (2003), 624 97-107.

The above-named article discloses compounds having the following formulae:

The article is hereby incorporated by reference in its entirety.

“Antileishmanial Activities of Several Classes of Aromatic Dications.” Brendle. James J., et al., Antimicrobial Agents and Chemotherapy (2002), 46(3), 797-807.

The above-named article discloses compounds having the following formulae:

The article is hereby incorporated by reference in its entirety.

PCT International Application No. WO 2000/010990 A2

The above-named PCT application describes compounds having the formula:

wherein each R is independently H, alkyl, alkoxy, or C(:NR⁵)NR⁵R⁶; R¹-R⁴=H, halo, alkyl, alkoxy, etc.; R⁵=H, alkyl, alkoxy, aryl, etc.; alternativey, R⁵R⁵=alkylene, etc.; R⁶=H, OH, alkyl, alkoxy, etc.; and Z=O or S.

PCT Application No. WO 2000/010990 A2 is hereby incorporated by reference in its entirety.

“Heterogeneity in The Actions of Drugs that Bind in The DNA Minor Groove.” Albert, Fred G., et al., Biochemistry (1999), 38(31), 10135-10146.

The above-named article describes the compound of formula:

The article is hereby incorporated by reference in its entirety.

“In Vitro Antifungal Activities of a Series of Dication-substituted Carbazoles, Furans, and Benzimidazoles,” Del Poeta, Maurizio, et al., Antimicrobial Agents and Chemotherapy (1998), 42(10), 2503-2510.

The above-named articled describes the compounds having the following formulae:

The article is hereby incorporated by reference in its entirety.

PCT International Application No. WO 96/40117 A1

The above-named PCT application describes compounds having the formula:

Wherein R=halo, alkyl, alkoxy, aryl, or C(:NR²)NR²R³; R¹=halo, alkyl, aryl, etc.; R²=H, alkyl, aryl, etc.; and R³=H, OH, alkyl, aryl, etc.

PCT Application No. WO 96/40117 A1 is hereby incorporated by reference in its entirety.

“Effect of new diamidines against Leishmania donovani infection,” Chauhan P. M. S., et al., Indian Journal of Experimental Biology (1993), 31(2), 196-8.

The above-named article describes the compounds having the following formulae:

The article is hereby incorporated by reference in its entirety.

“Investigations on mutagenicity and genotoxicity of pentamidine and some related trypanocidal diamidines.” Stauffert, I., et al., Mutation Research (1990), 245(2), 93-8.

The above-named article describes the compounds having the following formulae:

The article is hereby incorporated by reference in its entirety.

“Synthesis of 2,7-diamidinoxanthone, thioxanthone and related compounds as potential leishmanicides,” Chauhan, P. M. S., et al., Organic Chemistry Including Medicinal Chemistry (1987), 26B(3) 248-50.

The above-named article describes compounds having the formula:

wherein Z¹=S or O and Z²=O or is absent. The article is hereby incorporated by reference in its entirety.

“Inhibitory activity of diarylamidine derivatives on murine leukemia L1210 cell growth,” Balzarini, Jan, et al., Investigational New Drugs (1983), 1(2), 103-15.

The above-named article describes compounds having the formula:

wherein X=NH, O, S SO₂, or CH₂; Y=CH, CNH₂, N, etc.; R¹ and R²=amidino, imidazolino, etc.; Z=CH:CH, PhO, CONH, NH, etc.; and n=0 or 1.

In other embodiments, the above-named article describes compounds having the formula:

wherein R¹ and R²=amidino or imidazolino and Z=CH:CH, NHN:N, etc.

In other embodiments, the above-named article describes compounds having the formula:

wherein X=O, S or NH; Y=CH, CMe, N; and R¹ and R²=amidino or imidazolino.

In other embodiments, the above-named article describes compounds having the formula:

wherein X=NH; Y=CH; Z=CH:CH; R¹ and R²=imidazolino; and n=0 or 1.

The above-named article is hereby incorporated by reference in its entirety.

“Amino derivatives of 9H-fluorene,” Ferranti, A., et al., Farmaco, Edizione Scientifica (1982), 37(3), 199-204.

The above-named article describes compounds having the formula:

wherein X=O or is absent and R=R1=amidino; alternatively, X=O, R=amidino, CONH(CH₂)_(n)C(:NH)NH₂, R¹=H, and n=2 or 3. The article is hereby incorporated by reference in its entirety.

“Antifungal and antibacterial activities of diarylamidine derivatives,” Anne, Jozef, et al., Antimicrobial Agents and Chemotherapy (1980), 18(2), 231-9.

The above-named article describes compounds having the following formulae:

wherein R¹ and R²=C(:NH)NH₂, imidazolino, etc.; X, X¹, and X²=NH, O, S, etc.; Y=CH, CNH₂, CMe, or N; Z=CH:CH, NHN:N, C₆H₄O, NHCOC₆H₄CONH, etc. The article is hereby incorporated by reference in its entirety.

“Diaryl amidine derivatives as oncornaviral DNA polymerase inhibitors,” De Clercq, E., et al. Journal of Medicinal Chemistry (1980), 23(7), 787-95.

The above-named article describes the compounds having the following formulae:

The article is hereby incorporated by reference in its entirety.

Boykin, et al., J. Med. Chem. 41, 124-129 (1998)

The above-named article describes compounds having the formula:

wherein R is selected from the group consisting of H, Pr, i-Pr, c-Pr, c-pentyl, and i-amyl. The article is hereby incorporated by reference in its entirety.

Biochemistry, 40, 2511 (2001)

The above-named article describes the compound having the formula:

The article is hereby incorporated by reference in its entirety.

Other publications describing pentamidine analogs suitable for use as disclosed herein include: “2,4-Diphenyl Furan Diamidines as Novel Anti-Pneumocystis carinii Pneumonia Agents,” Francesconi, et al., J. Med. Chem. 42:2260-2265, 1999; “Trypanocidal Activity of Conformationally Restricted Pentamidine Congeners,” Donkor, et al., J. Med. Chem. 46:1041-1048 2003; “Antimicrobial Activity of the DNA Minor Groove Binders Furamidine and Analogs,” Boykin, J. Braz. Chem. Soc., 13(6):763-771, 2002; “Antileishmanial Activities of Several Classes of Aromatic Dications,” Brendle, et al., Antimicrobial Agents and Chemotherapy, 46(3):797-807 March 2002; “Comparative Efficacy Evaluation of Dicationic Carbazole Compounds, Nitazoxanide, and Paromomycin against Cryptosporidium parvum Infections in a Neonatal Mouse Model,” Blagburn, et al, Antimocrobial Agents and Chemotherapy, 42(11):2877-2882 November 1998; “Inhibitory effects of pentamidine analogues on protein biosynthesis in vitro,” Bielawski, et al., 47(1):113-120, 2000; “Amoebicidal Efficiencies of Various Diamidines against Two Strains of Acanthamoeba polyphaga,” Perrine, et al., Antimicrobial Agents and Chemotherapy, 39(2):339-342 February 1995; “Synthesis, and biological evaluation of new 1,3,4-thiadiazolium-2-phenylamine derivatives against Leishmania amazonensis promastigotes and amastigotes,” da Silva, et al., European Journal of Medicinal Chemistry, 37:979-984, 2002; “Effect of amidine derivatives on nitric oxide production by Leishmania amazonensis promastigotes and axenic amastigotes,” Genestra, et al., Nitric Oxide, 8:1-6, 2003; “Synthesis of Analogues of Pentamidine as Potential Anti-Pneumocystis Carinii Agents,” Huang, et la., Electronic Conference on Synthetic Organic Chemistry (ECSOC-5), http://www.mdpi.org/ecsoc-5.htm, September 2001; “Coordination chemistry of two-tricyclic bisamidines,” Widlicka, et al., Abstracts of Papers, 224^(th) ACS National Meeting, Boston, Mass., USA, Publisher: American Chemical Society, Washington D.C., Aug. 18-22, 2002; “Elongation factor 2 as a target for selective inhibition of protein synthesis in vitro by the novel aromatic bisamidine,” Gajko-Galicka, et al., Molecular and Cellular Biochemistry, 223(1&2):159-164, 2002; “Bisamidino benzofuran comounds as sodium/proton exchanger subtype 3 (NHE-3) inhibitors,” Gericke, et al., PCT International Application No. WO 2001/072742, A1, Oct. 4, 2001; DNA-binding properties and cytotoxicity of extended aromatic bisamidines in breast cancer MCF-7 cells,“Bielawski, et al., Polish Journal of Pharmacology, 53(2):143-147, 2001; “Aromatic extended bisamidines: synthesis, inhibition of topoisomerases, and anticancer cytotoxicity in vitro,” Archiv der Pharmazie (Weinheim, Germany), 334(7):235-240, 2001; “DNA-binding activity and cytotoxicity of the extended diphenylfuran bisamidines in breast cancer MCF-7 cells,” Bielawski, et al., Biological & Pharmaceutical Bulletin, 24(6):704-706, 2001; “Preparation of bis(aminoalkyl- or amidinophenoxy)arylene- and heteroatom-interrupted alkanes and analogs as tryptase inhibitors,” Anderskewitz, et al., German Patent Application No. DE 99-19955476, Nov. 18, 1999; “A COMFA study on antileishmaniasis bisamidines,” Montanari, et al., Molecular Modeling and Prediction of Bioactivity, Proceedings of the European Symposium on Quantitative Structure-Activity Relationships: Molecular Modeling and Prediction of Bioactivity, 12^(th), Copenhagen, Denmark, Aug. 23-28, 1998 (2000); “Bis-Cationic heteroaromatics as macrofilaricides: synthesis of bis-amidine and bis-guanylhydrazone derivatives of substituted Imidazo[1,2-a]pyridines,” Sundberg, et al., Journal of Medicinal Chemistry, 41(22):4317-4328, 1998; “Discovery of N-[2[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-difluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phenoxy]pyridine-4-yl]-N-methylglycine (ZK-807834): A Potent, Selective, and Orally Active Inhibitor of the Blood Coagulation Enzyme Factor Xa1,” Phillips, et al., Journal of Medicinal Chemistry, 41(19):3557-3562, 1998; “Active site-directed thrombin inhibitors. II. Studies related to arginine/guanidine bioisosteres,” St. Laurent, et al., Biooganic & Medicinal Chemistry, 3(8):1145-56, 1995; “Bis(5-amidino-2-benzimidazolyl)methane and related amidines are potent, reversible inhibitors of mast cell tryptases,” Caughey, et al., Journal of Pharmacology and Experimental Therapeutics, 264(2):676-82,1993; “Antiparasitic agents. Part VI. Synthesis of 1,2-, 1,3-, and 1,4-bis[4-substituted(aryloxy)]benzenes and their biological activities,” Schauhan, P. M., et al., Indian Journal of Chemistry, Section B: Organic Chemistry Including Medicinal Chemistry, 27B(1):38-42, 1988; “Synthesis and study of the antileukemic activity of N,N′-substituted amidines and bisamidines,” Dumont, et al., Journal de Pharmacie de Belgique, 40(6):373-86, 1985; “Antiprotozoal diamidines,” Edward A. Glazer, U.S. Pat. No. 4,546,113, Oct. 8, 1985; “Aromatic extended bisamidines: synthesis, inhibition of topoisomerases, and anticancer cytotoxicity in vitro,” Bielawski, K., et al., Archiv der Pharmazie, 334(7):235-40, July 2001; “Synthesis of antiproteolytically active 3,5-bis(4-amidinobenzyl)- and 3,5-bis(4-amidinobenzylidene)piperidone-(4) derivatives (author's transl.),” Richter, P., et al., Die Pharmazie, 35(2):75-77, February 1980; “Bisamidines of 2,6-diaminoanthraquinone as antiamebic agents,” Fabio, P. F., et al., Journal of Medicinal Chemistry, 21(3):273-6, March 1978; “Synthetic inhibitors of serine proteinases. 11. Report: The inhibition of trypsin, plasmin and thrombin by new bisamidino compounds,” Walsmann, P., et al., Acta biologica et medica Germanica, 35(2):K1-8, 1976; “Effects of compound Structure on Carbazole Dication-DNA Complexes: Tests of the Minor-Groove Complex Models,” Tanious, et al., Biochemistry, 39(39):12091-12101, 2000; “Synthesis and anti-Pneumocystis carinii pneumonia activity of novel dicationic dibenzothiophenes and orally active prodrugs,” Patrick, D. A., et al., European Journal of Medicinal Chemistry, 34(7&8):575-583, 1999; “Dicationic dibenzofuran derivatives as anti-Pneumocystis carinii pneumonia agents: synthesis, DNA binding affinity, and anti-P. carinii activity in an immunosuppressed rat model,” Wang, S., et al., European Journal of Medicinal Chemistry, 34(3):215-224, 1999; “Anti-Pneumocystis activities of aromatic diamidoxime prodrugs,” Hall, J. E., et al., Antimicrobial Agents and Chemotherapy, 42(3):666-674, 1998; “Anti-Pneumocystis carinii pneumonia activity of dicationic carbazoles,” Patrick, D. A., et al., European Journal of Medicinal Chemistry, 32(10):781-793, 1997; “Pharmacokinetic properties of antileukemic and trypanocidal compounds with amidino and imidazolinyl groups,” Gluth, W. P., et al., arzneimittel-Forschung, 34(11):1542-51, 1984; “Reduction of N-hydroxylated compounds: amidoximes (N-hydroxyamidines) as pro-drugs of amidines,” Clement Bernd Pharmaceutical Institute, Univ. of Kiel, Germany, Drug metabolism reviews, 34(3):565-79, August 2002; “Trypanocidal Activity of Conformationally Restricted Pentamidine Congeners,” Donkor, I. O., et al., Journal of Medicinal Chemistry, 46(6):1041-1048, 2003; “Trypanocidal activity of dicationic compounds related to pentamidine,” Donkor, I. O., et al., European Journal of Medicinal Chemistry, 36(6):531-538, 2001; “In vitro antimicrobial activity of aromatic diamidines and diimidazolines related to pentamidine,” Donkor, I. O., et al., European Journal of Medicinal Chemistry, 34(7&8):639-643, 1999; “Diarylamidines and -imidazolines as NMDA receptor antagonists,” Tao, B., et al.,. Book of Abstracts, 216^(th) ACS National Meeting, Boston, MEDI-111, Aug. 23-27, 1998; “Inhibitory effects of pentamidine analogs on spermine stimulated ligand binding to the NMDA receptor complex,” Donkor, I. O., et al., Bioorganic & Medicinal Chemistry Letters, 7(11):1455-1460, 1997; “Pentamidine congeners. 4. DNA binding affinity and anti-Pneumocystis carinii activity of butamidine analogs,” Donkor, I. O., et al., Bioorganic & Medicinal Chemistry Letters, 6(16):1967-70, 1996; “Structure-activity relationships of pentamidine analogs against Giardia lamblia and correlation of antigiardial activity with DNA-binding affinity,” Bell, C. A., et al., Antimicrobial Agents and Chemotherapy, 35(6):1099-107, 1991; “Structure-activity relationships of analogs of pentamidine against Plasmodium falciparum and Leishmania mexicana amazonensis,” Bell, C. A., et al., Antimicrobial Agents and Chemotherapy, 34(7):1381-6, 1990; “Analogs of 1,5-bis(4-amidinophenoxy)pentane (pentamidine) in the treatment of experimental Pneumocystis carinii pneumonia,” Tidwell, R. R., et al., Journal of Medicinal Chemistry, 33(4):1252-7, 1990; “Structure-activity relationships of pentamidine analogs against Giardia lamblia and correlation of antigiardial activity with DNA-binding affinity,” Bell, C. A., et al., Antimicrobial agents and chemotherapy, 35(6):1099-107, June 1991; “Structure-activity relationships of analogs of pentamidine against Plasmodium falciparum and Leishmania mexicana amazonensis,” Bell, C. A., et al., Antimicrobial agents and chemotherapy, 34(7):1381-6, July 1990; “Structure-activity studies of novel amidine analogues of chlorambucil: Correlation of cytotoxic activity with DNA-binding affinity and topoisomerase II inhibition,” Bielawska, A., et al., Archiv der Pharmazie (Weinheim, Germany), 336(6-7):293-299, 2003; “Synthesis and structure-activity relationships of novel parenteral carbapenems, CS-023 (R-115685) and related compounds containing an amidine moiety,” Kawamoto, I., et al., Journal of Antibiotics, 56(6):565-579, 2003; “Noncovalent tripeptidic thrombin inhibitors incorporating amidrazone, amine and amidine functions at P1,” Lee, K., et al., Bioorganic & Medicinal Chemistry Letters, 12(7):1017-1022, 2002; “Benzoyl and cinnamoyl nitrogen mustard derivatives of benzoheterocyclic analogues of the tallimustine: synthesis and antitumor activity,” Baraldi, P. G., et al., Biooragnic & Medicinal Chemistry 10(5):1611-1618, 2002; “Selective heterocyclic amidine inhibitors of human inducible nitric oxide synthase,” Moornann, A. E., et al., Bioorganic & Medicinal Chemistry Letters, 11(19):2651-2653, 2001; “Preparation of carbohydrate amidine derivatives as glycosidase inhibitors,” Sakata, K., et al., Japanese Patent No. JP 2001247589, A2, Sep. 11, 2001; “Development of serine protease inhibitors displaying a multicentered short (<2.3.ANG.) hydrogen bond binding mode: Inhibitors of urokinase-type plasminogen activator and factor Xa.,” Vemer, E., et al., Journal of Medicinal Chemistry, 44(17):2753-2771, 2001; “DNA-binding activity and cytotoxicity of the extended diphenylfuran bisamidines in breast cancer MCF-7 cells,” Bielawski, K., et al., Biological & Pharmaceutical Bulletin, 24(6):704-706, 2001; “Effect of amidine derivatives on Leishmania amazonensis axenic amastigotes. Preliminary studies of structure-activity relationships,” Canto-Cavalheiro, M. M., et al., Arzneimittel-Forschung, 50(10):925-928, 2000; “Rationally-designed guanidine and amidine fungicides,” Liebeschuetz, J. W., et al., Pesticide Science, 50(3):258-274, 1997; “Synthesis and biochemical activity of novel amidine derivatives as ml muscarinic receptor agonists,” Ojo, B., et al., Bioorganic & Medicinal Chemistry, 4(10):1605-1615, 1996; Bis(5-amidino-2-benzimidazolyl)methane and related amidines are potent, reversible inhibitors of mast cell tryptases,” Caughey, G. H., et al., Journal of Pharmacology and Experimental Therapeutics, 264(2):676-82, 1993; “Structure-activity relationships among amidine acaricides and insecticides,” Knowles, C. O., Insectic. Mode Action, pp. 243-77, 1982; “Structure-activity relations of amidine derivatives,” Fastier, F. N., Pharmacological Reviews, 14:37-90, 1962; “Structure-activity studies of novel amidine analogues of chlorambucil: correlation of cytotoxic activity with DNA-binding affinity and topoisomerase II inhibition,” Bielawska A., et al., Archiv der Pharmazie, 336(6-7):293-9, August 2003; “Development of serine protease inhibitors displaying a multicentered short (<2.3 A) hydrogen bond binding mode: inhibitors of urokinase-type plasminogen activator and factor Xa,” Verner E., et al., Journal of Medicinal Chemistry, 44(17):2753-71, 2001; “Synthesis, characterization, and structure-activity relationships of amidine-substituted (bis)benzylidene-cycloketone olefin isomers as potent and selective factor Xa inhibitors,” Guilford, W. J., et al., Journal of Medicinal Chemistry, 42(26):5415-25, Dec. 30, 1999; “On the structure-activity relationship of histamine H2-receptor antagonists based on the X-ray crystal structures and 1H-NMR spectra of amidine derivatives,” Ishida, T., et al., Molecular Pharmacology, 31(4):410-6, April 1987; “Structure-activity relationships in distamycin A analogues: effect of alkyl groups on the pyrrole nitrogen at the non-amidine end of the molecule combined with methyl elimination in the following ring,” Grehn L., et al., Acta chemica Scandinavica. Series B: Organic chemistry and biochemistry, 40(2):145-51, February 1986; “Quantitative structure-activity relationships and molecular graphics in ligand receptor interactions: amidine inhibition of trypsin,” Recanatini M., et al., Moelcular Pharmacology, 29(4):436-46, April 1986; “Structure-activity relationships of pyrrole amidine antiviral antibiotics III: preparation of distamycin and congocidine derivatives based on 2,5-disubstituted pyrroles,” Bialer, M., et al., Journal of Pharmaceutical Sciences, 69(11):1334-8, November 1980; “Structure-activity relationships of pyrrole amidine antiviral antibiotics. 1. Modifications of the alkylamidine side chain,” Bialer, M., et al., Journal of Medicinal Chemistry, 22(11):1296-301, November 1979; “Oximes, amidoximes and hydroxamic acids as nitric oxide donors,” Koikov, L. N., et al., Mendeleev Communications, (4):165-168, 1998; “Anti-Pneumocystis activities of aromatic diamidoxime prodrugs,” Hall, J. E., et al., Antimicrobial Agents and Chemotherapy, 42(3):666-674, 1998; “Hemodynamic effects of a series of new trypanocidal indoleamidino compounds,” Steinmann, U., et al., Drug Development Research, 7(2):153-63, 1986; “Hydroxylamine derivatives as potential antimalarial agents. 2. Hydroxamates and amidoximes,” Hynes, J. B., et al., Journal of Medicinal Chemistry, 15(11):1194-6, 1972; and “Cyclopolycondensations. IX. Syntheses of fully polyetherpoly(1,2,4-oxadiazoles),” Dogoshi, N., et al., Makromolekulare Chemie, 108:170-81, 1967; all of which are hereby incorporated by reference in their entirety.

Microbial Species

The microbial species to be inhibited through the use of an efflux pump inhibitor as described herein, can be from multiple bacterial groups or species. Non-limiting examples include one or more of the following: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, or Staphylococcus saccharolyticus.

A particularly appropriate example of a microbe appropriate for the use of an efflux pump inhibitor disclosed herein is a pathogenic bacterial species, Pseudomonas aeruginosa, which is intrinsically resistant to many of the commonly used antibacterial agents. Exposing this bacterium to an efflux pump inhibitor can significantly slow the export of an antibacterial agent from the interior of the cell or the export of siderophores. Therefore, if an antibacterial agent is administered in conjunction with the efflux pump inhibitor, the antibacterial agent, which would otherwise be maintained at a very low intracellular concentration by the export process, can accumulate to a concentration that will inhibit the growth of the bacterial cells. This growth inhibition can be due to either bacteriostatic or bactericidal activity, depending on the specific antibacterial agent used. While P. aeruginosa is an example of an appropriate bacterium, other bacterial and microbial species may contain similar broad substrate pumps, which actively export a variety of antimicrobial agents, and thus can also be appropriate targets.

Antimicrobial Agents

In particular embodiments various antibacterial agents can be used in combination with the efflux pump inhibitors described herein. These include quinolones, tetracyclines, glycopeptides, aminoglycosides, β-lactams, rifamycins, macrolides/ketolides, oxazolidinones, coumermycins, chloramphenicol, and glycylcycline. In particular embodiments, an antibiotic of the above classes can be, for example, one of the following:

Beta-Lactam Antibiotics

imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763

Macrolides

azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin, roxithromycin, troleandomycin

Ketolides

telithromycin, cethromycin

Quinolones

amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin; gemifloxacin; garenoxacin; PD131628, PD138312, PD140248, Q-35, AM-1155, NM394, T-3761, rufloxacin, OPC-17116, sitafloxacin (Sato, K. et al., 1992, Antimicrob Agents Chemother. 37:1491-98), DV-7751a (Tanaka, M. et al., 1992, Antimicrob. Agents Chemother. 37:2212-18), and (Kurosaka et al., Interscience Conference on Antimicrobial Agents and Chemotherapy, 2003, 43rd:Chicago (F-1061)).

Tetracyclines, Glycylcyclines and Oxazolidinones

chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline; linezolide, eperozolid

Aminoglycosides

amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin

Lincosamides

clindamycin, lincomycin.

Methods of Treatment or Prophylaxis

It has been discovered that pentamidine, which is known to inhibit growth of various protozoal pathogens, such as Pneumocystis, Trypanosoma and Leishmania, also is capable of inhibiting cellular efflux pumps of bacteria or other microbes. Such efflux pumps export substrate molecules from the cytoplasm in an energy-dependent manner, and the exported substrate molecules can include antibacterial agents. Such efflux pump inhibitors are useful, for example, for treating microbial infections by reducing the export of a co-administered antimicrobial agent or by preventing the export of a compound synthesized by microbes (e.g., bacteria) to allow or improve their growth. While the endogenous substrates of efflux pumps are not yet identified, there are some indications that efflux pumps may be important for bacterial virulence. Thus, also disclosed herein are compositions that include such efflux pump inhibitors and methods for treating microbial infections using those compositions.

In some embodiments, a method is provided for treating a microbial infection in an animal, specifically including in a mammal, by treating an animal suffering from such an infection with an antimicrobial agent and an efflux pump inhibitor, which increase the susceptibility of the microbe for that antimicrobial agent. Such efflux pump inhibitors can be selected from any of the pentamidine or pentamidine analog compounds generically or specifically described herein. In this way a microbe involved in the infection can be treated using the antimicrobial agent in smaller quantities, or can be treated with an antimicrobial agent, which is not therapeutically effective when used in the absence of the efflux pump inhibitor. Thus, this method of treatment is especially appropriate for the treatment of infections involving microbial strains that are difficult to treat using an antimicrobial agent alone due to a need for high dosage levels (which can cause undesirable side effects), or due to lack of any clinically effective antimicrobial agents. However, it is also appropriate for treating infections involving microbes that are susceptible to particular antimicrobial agents as a way to reduce the dosage of those particular agents. This can reduce the risk of side effects. It is also appropriate for treating infections involving microbes that are susceptible to particular antimicrobial agents as a way of reducing the frequency of selection of resistant microbes. In particular embodiments the microbe is a bacterium, which may, for example, be from any of the groups or species indicated above.

In some embodiments, a method is provided for prophylactic treatment of a mammal. In this method, an antimicrobial agent and an efflux pump inhibitor is administered to a mammal at risk of a microbial infection, e.g., a bacterial infection. The efflux pump inhibitor can be selected from any of the pentamidine or pentamidine analog compounds generically or specifically described herein.

In some embodiments, a method is provided for enhancing the antimicrobial activity of an antimicrobial agent against a microbe, in which such a microbe is contacted with an efflux pump inhibitor, and an antibacterial agent. The efflux pump inhibitor can be selected from any of the pentamidine or pentamidine analog compounds generically or specifically described herein. In one embodiment, the efflux pump inhibitor is pentamidine. Thus, this method makes an antimicrobial agent more effective against a cell, which expresses an efflux pump when the cell is treated with the combination of an antimicrobial agent and an efflux pump inhibitor. In particular embodiments the microbe is a bacterium or a fungus, such as any of those indicated above; the antibacterial agent can be selected from a number of structural classes of antibiotics including, e.g., beta-lactams, glycopeptides, aminoglycosides, quinolones, oxazolidinones, tetracyclines, rifamycins, coumermycins, macrolides, and chloramphenicol. In particular embodiments an antibiotic of the above classes can be as stated above.

In other embodiments, a method is provided for suppressing growth of a microbe, e.g., a bacterium, expressing a multidrug resistance efflux pump. As illustrated by the case where the microbe is a bacterium, the method involves contacting that bacterium with an efflux pump inhibitor, in the presence of a concentration of antibacterial agent below the MIC of the bacterium. The efflux pump inhibitor can be selected from any of the pentamidine or pentamidine analog compounds generically or specifically described herein. This method is useful, for example, to prevent or cure contamination of a cell culture by a bacterium possessing an efflux pump. However, it applies to any situation where such growth suppression is desirable.

In some embodiments, any of the compounds generically or specifically described herein may be administered as an efflux pump inhibitor either alone or, in conjunction with another therapeutic agent. In some embodiments, any of the compounds generically or specifically described herein may be administered as an efflux pump inhibitor in conjunction with any of the antimicrobial agents specifically or generically described herein, as well as with any other antimicrobial agent useful against the species of microbe to be treated, when such microbe do not utilize an efflux pump resistance mechanism. In some embodiments, the antimicrobial agents are administered at their usual recommended dosages. In other embodiments, the antimicrobial agents are administered at reduced dosages, as determined by a physician. For all conventional antimicrobials on the market, and many in clinical development, dosage ranges and preferred routes of administration are well established, and those dosages and routes can be used in conjunction with the efflux pump inhibitors of the present invention. Reduced dosages of the antimicrobials are contemplated due to the increased efficacy of the antimicrobial when combined with an efflux pump inhibitor.

In some embodiments, a compound disclosed herein is administered to a pulmonary site of infection, such as through inhalation of an aerosol. In some embodiments, the pulmonary infection is a Pseudomonas infection. In various embodiments, one or more administrations of a compound disclosed herein is provided so as to achieve a delivered daily dose of at least about 5 mg, 10 mg, 15 mg, 40 mg, 60 mg, 80 mg, or 100 mg.

In some embodiments, a compound disclosed herein is administered along with an antimicrobial agent. The two agents may be administered in a predetermined ratio. For example, the agents may be administered in a 1:1 ratio, 1:2 ratio, 2:1 ratio, etc. The agents may be administered separately, together, simultaneously, or sequentially. The agents may be administered as a combined, fixed dosage form or as separate dosage forms.

In some embodiments, a subject is identified as infected with bacteria that are resistant to an antimicrobial agent. The subject may then be treated with the antimicrobial agent in combination with a compound disclosed herein. A subject may be identified as infected with bacteria that are resistant based on observing an ineffective response of the infection to the antimicrobial. Alternatively, the bacteria may be cultured and identified as a known resistant strain by appropriate microbiological techniques known in the art.

In some embodiments, a subject is identified as a subject that is infected with bacteria that are capable of developing resistance to an antimicrobial. The subject may then be treated with the antimicrobial agent in combination with a compound disclosed herein. A subject may be identified as infected with bacteria that are capable of developing resistance by diagnosing the subject as having symptoms that are characteristic a bacterial infection with a bacteria species known to have resistant strains or a with a bacteria that is a member of group that are known to have resistant strains. Alternatively, the bacteria may be cultured and identified as a species known to have resistant strains or a bacteria that is a member of group that are known to have resistant strains.

In some embodiments, an efflux pump inhibitor is administered at a level sufficient to overcome or suppress the emergence of efflux pump-mediated resistance in bacteria. In some embodiments, this level produces the effective efflux pump inhibitory concentration at the site of infection. In other embodiments, this level produces an effect equivalent to shutting down all efflux pumps in the bacteria.

In some embodiments, a subject is identified as a subject that is at risk of infection with bacteria. The subject may then be prophylactically treated with an efflux pump inhibitor and an antimicrobial agent in order to prevent infection with a resistant bacterial strain. For example, subjects in environments likely to have resistant bacteria, such as a hospital, may be prophylactically treated.

In some embodiments, a subject is treated with an efflux pump inhibitor that is not otherwise generally effective as an antimicrobial. Thus, for example, the MIC of the efflux pump inhibitor may be greater than about 32 μg/ml, 64 μg/ml, 128 μg/ml, or 256 μg/ml.

In some embodiments, the subject to which an efflux pump inhibitor is administered is a human. In other embodiments, the subject is a non-human vertebrate. In another embodiments, the subject is a non-mammal mammal, bird, fish, amphibian, or reptile.

Screening for Efflux Pump Inhibitors

Potential efflux pump inhibitor compounds can be tested for their ability to inhibit multi-drug resistance efflux pumps of various microbes and to potentiate various antimicrobial agents by using the methods described herein as well as those known in the art. For example, strains of microbes known to overexpress efflux pumps may be treated with the antimicrobial agent with and without the test efflux pump inhibitor compound. A checkerboard assay may be used with varying concentrations of both antimicrobial agent and test compound to determine the relative concentrations at which potentiation is observed.

In one non-limiting example, treatment of P. aeruginosa with a test compound allows obtaining one or more of the following biological effects:

-   -   1) P. aeruginosa strains will become susceptible to antibiotics         that could not be used for treatment of pseudomonas infections,         or become more susceptible to antibiotics, become more         susceptible to antibiotics currently used for treatment of         pseudomonas infections.     -   2) P. aeruginosa strains which developed resistance to         antibiotics currently used for treatment of pseudomonas         infections will become susceptible to these antibiotics.     -   3) Inhibition of the pump will result in a decreased frequency         of resistance development to antibiotic, which is a substrate of         the pump.

Obtaining even one of these effects provides a potential therapeutic treatment for infections by this bacterium. Also, similar pumps are found in other microorganisms. Some or all of the above effects can also be obtained with those microbes, and they are therefore also appropriate targets for detecting or using efflux pump inhibitors.

Pharmaceutical Compositions

For purposes of co-administration of an EPI as described herein and another antimicrobial compound, the EPI may be administered by the same route as the other anti-bacterial compound, either simultaneously or sequentially. For example, both the EPI and the anti-bacterial compound may be administered using an inhaler. In such a case, both compounds may be administered simultaneously by having them both present in the same compartment such that they are aerosolized togeth er. Alternatively, the compounds may be contained in separate compartments and mixed in the process of inhaler activation, such as described in PCT Publication No. WO 00/64520, which is hereby incorporated by reference in its entirety. Another embodiment comprises separate drug compartments in the same inhaler that may activated sequentially, such as described in U.S. Pat. No. 6,523,536, which is hereby incorporated by reference in its entirety.

In other embodiments, the EPI and other anti-bacterial compound are both administered i.v., either mixed in a fixed drug formulation or present in separate formulations. In other embodiments, the EPI and other anti-bacterial compound are both administered orally, either in the same fixed formulation or in separate formulations. In still other embodiments, the EPI and other anti-bacterial compound are both administered i.m., again either mixed in a fixed drug formulation or present in separate formulations.

In some embodiments, the EPI and other anti-bacterial compound to be co-administered are administered by separate routes. For example, the EPI may be administered by inhalation while the other anti-bacterial compound is administered i.v., i.m., or orally. Any other possible combination of separate route administration is also contemplated.

In some embodiments, an efflux pump inhibitor disclose herein is combined in a fixed combination with an antimicrobial agent. In some embodiments, the fixed combination includes the efflux pump inhibitor and antimicrobial agent packaged in separate containers, such as in a multi-chamber inhaler. In other embodiments, the fixed combination includes the efflux pump inhibitor and antimicrobial agent being physically combined together in the same formulation (e.g., a combined, fixed dosage form).

Administration

The efflux pump inhibitors disclosed herein may be administered at a therapeutically effective dosage, e.g., a dosage sufficient to provide treatment for the disease states previously described. While human dosage levels have yet to be optimized for the compounds of the invention, generally, a daily dose of pentamidine and for most of the inhibitors described herein is from about 0.05 to 100 mg/kg of body weight, preferably about 0.10 to 10.0 mg/kg of body weight, and most preferably about 0.15 to 1.0 mg/kg of body weight. Thus, for administration to a 70 kg person, the dosage range would be about 3.5 to 7000 mg per day, preferably about 7.0 to 700.0 mg per day, and most preferably about 10.0 to 100.0 mg per day. The amount of active compound administered will, of course, be dependent on the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician; for example, a likely dose range for oral administration would be about 70 to 700 mg per day, whereas for intravenous administration a likely dose range would be about 700 to 7000 mg per day, the active agents being selected for longer or shorter plasma half-lives, respectively. Screening techniques described herein for pentamidine can be used with other efflux pump inhibitors described herein to establish the efficacy of those inhibitors in comparison to pentamidine, and the dosage of the inhibitor can thus be adjusted to achieve an equipotent dose to the dosages of pentamidine.

Administration of the compounds disclosed herein or the pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, orally, subcutaneously, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly. Oral and parenteral administration are customary in treating the indications that are the subject of the present invention.

Pharmaceutically acceptable compositions include solid, semi-solid, liquid and aerosol dosage forms, such as, e.g., tablets, capsules, powders, liquids, suspensions, suppositories, aerosols or the like. The compounds can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate. Preferably, the compositions are provided in unit dosage forms suitable for single administration of a precise dose.

The compounds can be administered either alone or more typically in combination with a conventional pharmaceutical carrier, excipient or the like (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like). If desired, the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like). Generally, depending on the intended mode of administration, the pharmaceutical formulation will contain about 0.005% to 95%, preferably about 0.5% to 50% by weight of a compound of the invention. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

In addition, the compounds can be co-administered with, and the pharmaceutical compositions can include, other medicinal agents, pharmaceutical agents, adjuvants, and the like. Suitable additional active agents include, for example, antimicrobial agents as described above. When used, other active agents may be administered before, concurrently, or after administration of an efflux pump inhibitor of the present invention. In some embodiments, an efflux pump inhibitor is co-administered with one or more other antimicrobial agents.

Thus, in the present invention, an efflux pump inhibitor compound as set forth herein can be administered through a first route of administration, and the antimicrobial agent can be administered through a second route. Thus, for example, an efflux pump inhibitor can be administered via a pulmonary route, e.g., through a nebulizer, atomizer, mister, aerosol, dry powder inhaler, or other suitable device or technique, and the antimicrobial can be administered via the same or a different route, e.g., orally, parenterally, intramuscularly, intraperitoneally, intratracheally, intravenously, subcutaneously, transdermally, or as a rectal or vaginal suppository. The blood levels of drugs are affected by the route of administration. Thus, in one preferred embodiment, when the efflux pump inhibitor is administered by a first route, and the antibiotic or antimicrobial through a second route, the dosages or dosage forms are adjusted, as appropriate, to match the pharmcokinetic profiles of each drug. This may also be done when both drugs are administered by the same route. In either event, conventional techniques, including controlled release formulations, timing of administration, use of pumps and depots, and/or use of biodegradable or bioerodible carriers can be used to match the pharmacokinetics of the two active moieties.

In one preferred embodiment, the compositions will take the form of a unit dosage form such as a pill or tablet and thus the composition may contain, along with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, or the like; a lubricant such as magnesium stearate or the like; and a binder such as starch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or the like. In another solid dosage form, a powder, marume, solution or suspension (e.g., in propylene carbonate, vegetable oils or triglycerides) is encapsulated in a gelatin capsule. Unit dosage forms in which the two active ingredients (inhibitor and antimicrobial) are physically separated are also contemplated; e.g., capsules with granules of each drug; two-layer tablets; two-compartment gel caps, etc.

Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as defined above and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection. The percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject. However, percentages of active ingredient of 0.01% to 10% in solution are employable, and will be higher if the composition is a solid, which will be subsequently diluted to the above percentages. In some embodiments, the composition will comprise 0.2-2% of the active agent in solution.

Aerosol Delivery

Efflux pump inhibitors (EPIs) as described herein, including any of the compounds generically or specifically described herein, can also be administered to the respiratory tract as an aerosol. In some embodiments, aersol delivery is used to treat an infection in the lungs, such as a Pseudomonas lung infection. Thus, for example, an efflux pump inhibitor may be administered directly to the site of infection through inhalation. Co-administered antimicrobial agents may also be administered through inhalation or through a systemic route of administration.

Several device technologies exist to deliver either dry powder or liquid aerosolized products. Dry powder formulations generally require less time for drug administration, yet longer and more expensive development efforts. Conversely, liquid formulations have historically suffered from longer administration times; yet have the advantage of shorter and less expensive development efforts. Pentamidine has previously been administered in a liquid aerosolized form. Pentamidine and the analogs disclosed herein are generally soluble and stable, however, the slow rate of pentamdine drug delivery through liquid aerosolized inhalation has contributed to acute intolerability. Furthermore, the previously used nebulizers for pentamidine delivery produces a particle size and distribution that does not provide maximum drug deposition at a bacterial site of infection.

Accordingly, in one embodiment, a particular formulation of efflux pump inhibitor disclosed herein is combined with a particular aerosolizing device to provide an aerosol for inhalation that is optimized for maximum drug deposition at a site of infection and minimal intolerability. Factors that can be optimized include solution or solid particle formulation, rate of delivery, and particle size and distribution produced by the aerosolizing device.

Particle Size and Distribution

Generally, inhaled particles are subject to deposition by one of two mechanisms: impaction, which usually predominates for larger particles, and sedimentation, which is prevalent for smaller particles. Impaction occurs when the momentum of an inhaled particle is large enough that the particle does not follow the air stream and encounters a physiological surface. In contrast, sedimentation occurs primarily in the deep lung when very small particles which have traveled with the inhaled air stream encounter physiological surfaces as a result of random diffusion within the air stream.

For intranasally administered drug compounds which are inhaled through the nose, it may be desirable for the drug to impact directly on the nasal mucosa; thus, large (ca. 5 to 100 micron) particles or droplets are generally preferred for targeting of nasal delivery. Pulmonary drug delivery may be accomplished by inhalation of an aerosol through the mouth and throat. Particles having a mass median aerodynamic diameter (MMAD) of greater than about 5 microns generally do not reach the lung; instead, they tend to impact the back of the throat and are swallowed and possibly orally absorbed. Particles having diameters of about 2 about 5 microns are small enough to reach the upper- to mid-pulmonary region (conducting airways), but are too large to reach the alveoli. Even smaller particles, i.e., about 0.5 to about 2 microns, are capable of reaching the alveolar region. Particles having diameters smaller than about 0.5 microns can also be deposited in the alveolar region by sedimentation, although very small particles may be exhaled.

In some embodiments, the particle size of the aerosol is optimized to maximize EPI deposition at the site of infection and to minimize intolerability. Aerosol particle size may be expressed in terms of the mass median aerodynamic diameter (MMAD). Large particles (e.g., MMAD>5 μm) may deposit in the upper airway because they are too large to navigate the curvature of the upper airway. Small particles (e.g., MMAD<2 μm) may be poorly deposited in the lower airways and thus become exhaled, providing additional opportunity for upper airway deposition. Hence, intolerability (e.g., cough and bronchospasm) may occur from upper airway deposition from both inhalation impaction of large particles and settling of small particles during repeated inhalation and expiration. Thus, in one embodiment, an optimum particle size is used (e.g., MMAD=2-4 μm) in order to maximize deposition at a mid-lung site of infection and to minimize intoleratiblity associated with upper airway deposition. Moreover, generation of a defined particle size with limited geometric standard deviation (GSD) may optimize deposition and tolerability. Narrow GSD limits the number of particles outside the desired MMAD size range. In one embodiment, an aerosol containing one or more compounds disclosed herein is provided having a MMAD from about 2 microns to about 4 microns with a GSD of less than or equal to about 2 microns. In another embodiment, an aerosol having an MMAD from about 2.8 microns to about 3.2 microns with a GSD from about 1.5 microns to about 1.8 microns is provided.

In some embodiments, aerosols intended for delivery to the nasal mucosa are provided for inhalation through the nose. For optimal delivery to the nasal cavities, inhaled particle sizes of about 5 to about 100 microns are useful, with particle sizes of about 30 to about 60 microns being preferred. For nasal delivery, a larger inhaled particle size is desired to maximize impaction on the nasal mucosa and to minimize or prevent pulmonary deposition of the administered formulation. Inhaled particles may be defined as liquid droplets containing dissolved drug, liquid droplets containing suspended drug particles (in cases where the drug is insoluble in the suspending medium), dry particles of pure drug substance, aggregates of drug nanoparticles, or dry particles of a diluent which contain embedded drug nanoparticles.

Efflux pump inhibitors disclosed herein (in the presence or absence of antibiotic) intended for respiratory delivery (either systemic or local) can be administered as aqueous formulations, as suspensions or solutions in halogenated hydrocarbon propellants, or as dry powders. Aqueous formulations may be aerosolized by liquid nebulizers employing either hydraulic or ultrasonic atomization. Propellant-based systems may use suitable pressurized metered-dose inhalers (pMDIs). Dry powders may use dry powder inhaler devices (DPIs), which are capable of dispersing the drug substance effectively. A desired particle size and distribution may be obtained by choosing an appropriate device.

Liquid Nebulizer

In one embodiment, a nebulizer is selected on the basis of allowing the formation of an aerosol of an efflux pump inhibitor disclosed herein in the presence or absence of antibiotic having a MMAD predominantly between 2 to 4 μm. In one embodiment, the delivered amount of efflux pump inhibitor in the presence or absence of antibiotic is efficacious for treatment and prophylaxis of respiratory infections. When the aerosol contains a large number of particles with a MMAD larger than 5 μm, these are deposited in the upper airways decreasing the amount of EPI or EPI-antibiotic combination delivered to the site of infection.

Previously, two types of nebulizers, jet and ultrasonic, have been shown to be able to produce and deliver aerosol particles having sizes between 2 and 4 μm. These particle sizes have been shown as being optimal for treatment of pulmonary bacterial infection cause by gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, K. oxytoca, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens, Haemophilus influenzae, Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and multidrug resistant Pseudomonas aeruginosa. However, unless a specially formulated solution is used, these nebulizers typically need larger volumes to administer sufficient amount of drug to obtain a therapeutic effect. A jet nebulizer utilizes air pressure breakage of an aqueous aztreonam solution into aerosol droplets. An ultrasonic nebulizer utilizes shearing of the aqueous aztreonam solution by a piezoelectric crystal. Typically, however, the jet nebulizers are only about 10% efficient under clinical conditions, while the ultrasonic nebulizer is only about 5% efficient. The amount of pharmaceutical deposited and absorbed in the lungs is thus a fraction of the 10% in spite of the large amounts of the drug placed in the nebulizer.

Accordingly, in one embodiment, a vibrating mesh nebulizer is used to deliver an aerosol of the efflux pump inhibitors disclosed herein. A vibrating mesh nebulizer consists of a liquid storage container in fluid contact with a diaphragm and inhalation and exhalation valves. In one embodiment, 1 to 5 ml of an efflux pump inhibitor formulation, with or without an antimicrobial agent is placed in the storage container and the aerosol generator is engaged producing atomized aerosol of particle sizes selectively between 1 and 4 μm.

By non-limiting example, an efflux pum inhibitor disclosed herein in the presence or absence of an antibiotic solution is placed in a liquid nebulization inhaler and prepared in dosages from 1-100 mg in 1-5 ml, preferably from 10-60 mg in 1-5 ml with MMAD particles sizes between 2 and 4 μm being produced.

For aqueous and other non-pressurized liquid systems, a variety of nebulizers (including small volume nebulizers) are available to aerosolize the formulations. Compressor-driven nebulizers incorporate jet technology and use compressed air to generate the liquid aerosol. Such devices are commercially available from, for example, Healthdyne Technologies, Inc.; Invacare, Inc.; Mountain Medical Equipment, Inc.; Pari Respiratory, Inc.; Mada Medical, Inc.; Puritan-Bennet; Schuco, Inc., DeVilbiss Health Care, Inc.; and Hospitak, Inc. Ultrasonic nebulizers rely on mechanical energy in the form of vibration of a piezoelectric crystal to generate respirable liquid droplets and are commercially available from, for example, Omron Heathcare, Inc. and DeVilbiss Health Care, Inc. Vibrating mesh nebulizers rely upon either piezoelectric or mechanical pulses to respirable liquid droplets generate. Other examples of nebulizers for use with the EPIs described herein are described in U.S. Pat. Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929; 4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304; 6,338,443; 5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575; 6,192,876; 6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,612,303; 6,644,304; 6,660,249; 5,549,102; 6,083,922; 6,161,536; 6,264,922; 6,557,549; 6,612,303; and 6,660,249 all of which are hereby incorporated by reference in their entirety. Commercial examples of nebulizers that can be used with the EPIs described herein include Aeroneb®, Aeroneb® Pro, and Aeroneb® Go produced by Aerogen; AERx® and AERx Essence™ produced by Aradigm; Porta-Neb®, Freeway Freedom™, Sidestream,, Ventstream and I-neb produced by Respironics, Inc.; and PARI LC-Plus®, PARI LC-Star®, and e-Flow produced by PARI, GmbH. By further non-limiting example, U.S. Pat. No. 6,196,219, is hereby incorporated by reference in its entirety.

In some embodiments, the drug solution is formed prior to use of the nebulizer by a patient. In other embodiments, the drug is stored in the nebulizer in solid form. In this case, the solution is mixed upon activation of the nebulizer, such as described in U.S. Pat. No. 6,427,682 and PCT Publication No. WO 03/035030, both of which are hereby incorporated by reference in their entirety. In these nebulizers, the solid drug, optionally combined with excipients to form a solid composition, is stored in a separate compartment from a liquid solvent.

The liquid solvent is capable of dissolving the solid composition to form a liquid composition, which can be aerosolized and inhaled. Such capability is, among other factors, a function of the selected amount and, potentially, the composition of the liquid. To allow easy handling and reproducible dosing, the sterile aqueous liquid may be able to dissolve the solid composition within a short period of time, possibly under gentle shaking. In some embodiments, the final liquid is ready to use after no longer than about 30 seconds. In some cases, the solid composition is dissolved within about 20 seconds, and advantageously, within about 10 seconds. As used herein, the terms “dissolve(d)”, “dissolving”, and “dissolution” refer to the disintegration of the solid composition and the release, i.e. the dissolution, of the active compound. As a result of dissolving the solid composition with the liquid solvent a liquid composition is formed in which the active compound is contained in the dissolved state. As used herein, the active compound is in the dissolved state when at least about 90 wt.-% are dissolved, and more preferably when at least about 95 wt.-% are dissolved. To measure disintegration and/or dissolution times, standard pharmacopoeial methods may be used. However, the methods must be selected to be appropriate for the specific form in which the solid composition is supplied. For instance, if the solid composition is a powder, it may be meaningless to measure disintegration. In other cases, an official method for measuring the dissolution time of the drug may not be relevant to the actual use of the nebulizer. In these cases, it may be better to determine the dissolution time under conditions that resemble those actually present when the nebulizer is used.

With regard to basic separated-compartment nebulizer design, it primarily depends on the specific application whether it is more useful to accommodate the aqueous liquid and the solid composition within separate chambers of the same container or primary package, or whether they should be provided in separate containers. If separate containers are used, these are provided as a set within the same secondary package. The use of separate containers is especially preferred for nebulizers containing two or more doses of the active compound. There is no limit to the total number of containers provided in a multi-dose kit. In one embodiment, the solid composition is provided as unit doses within multiple containers or within multiple chambers of a container, whereas the liquid solvent is provided within one chamber or container. In this case, a favorable design provides the liquid in a metered-dose dispenser, which may consist of a glass or plastic bottle closed with a dispensing device, such as a mechanical pump for metering the liquid. For instance, one actuation of the pumping mechanism may dispense the exact amount of liquid for dissolving one dose unit of the solid composition.

In another embodiment for multiple-dose separated-compartment nebulizers, both the solid composition and the liquid solvent are provided as matched unit doses within multiple containers or within multiple chambers of a container. For instance, two-chambered containers can be used to hold one unit of the solid composition in one of the chambers and one unit of liquid in the other. As used herein, one unit is defined by the amount of drug present in the solid composition, which is one unit dose. Such two-chambered containers may, however, also be used advantageously for nebulizers containing only one single drug dose.

In one embodiment of a separated-compartment nebulizer, a blister pack having two blisters is used, the blisters representing the chambers for containing the solid composition and the liquid solvent in matched quantities for preparing a dose unit of the final liquid composition. As used herein, a blister pack represents a thermoformed or pressure-formed primary packaging unit, most likely comprising a polymeric packaging material that optionally includes a metal foil, such as aluminum. The blister pack may be shaped to allow easy dispensing of the contents. For instance, one side of the pack may be tapered or have a tapered portion or region through which the content is dispensable into another vessel upon opening the blister pack at the tapered end. The tapered end may represent a tip.

In some embodiments, the two chambers of the blister pack are connected by a channel, the channel being adapted to direct fluid from the blister containing the liquid solvent to the blister containing the solid composition. During storage, the channel is closed with a seal. In this sense, a seal is any structure that prevents the liquid solvent from contacting the solid composition. The seal is preferably breakable or removable; breaking or removing the seal when the nebulizer is to be used will allow the liquid solvent to enter the other chamber and dissolve the solid composition. The dissolution process may be improved by shaking the blister pack. Thus, the final liquid composition for inhalation is obtained, the liquid being present in one or both of the chambers of the pack connected by the channel, depending on how the pack is held.

According to another embodiment, one of the chambers, preferably the one that is closer to the tapered portion of the blister pack, communicates with a second channel, the channel extending from the chamber to a distal position of the tapered portion. During storage, this second channel does not communicate with the outside of the pack but is closed in an air-tight fashion. Optionally, the distal end of the second channel is closed by a breakable or removable cap or closure, which may e.g. be a twist-off cap, a break-off cap, or a cut-off cap.

The solid composition itself can be provided in various different types of dosage forms, depending on the physicochemical properties of the drug, the desired dissolution rate, cost considerations, and other criteria. In one of the embodiments, the solid composition is a single unit. This implies that one unit dose of the drug is comprised in a single, physically shaped solid form or article. In other words, the solid composition is coherent, which is in contrast to a multiple unit dosage form, in which the units are incoherent.

Examples of single units which may be used as dosage forms for the solid composition include tablets, such as compressed tablets, film-like units, foil-like units, wafers, lyophilized matrix units, and the like. In a preferred embodiment, the solid composition is a highly porous lyophilized form. Such lyophilizates, sometimes also called wafers or lyophilized tablets, are particularly useful for their rapid disintegration, which also enables the rapid dissolution of the active compound.

On the other hand, for some applications the solid composition may also be formed as a multiple unit dosage form as defined above. Examples of multiple units are powders, granules, microparticles, pellets, beads, lyophilized powders, and the like. In one embodiment, the solid composition is a lyophilized powder. Such a dispersed lyophilized system comprises a multitude of powder particles, and due to the lyophilization process used in the formation of the powder, each particle has an irregular, porous microstructure through which the powder is capable of absorbing water very rapidly, resulting in quick dissolution.

Another type of multiparticulate system which is also capable of achieving rapid drug dissolution is that of powders, granules, or pellets from water-soluble excipients which are coated with the drug, so that the drug is located at the outer surface of the individual particles. In this type of system, the water-soluble low molecular weight excipient is useful for preparing the cores of such coated particles, which can be subsequently coated with a coating composition comprising the drug and, preferably, one or more additional excipients, such as a binder, a pore former, a saccharide, a sugar alcohol, a film-forming polymer, a plasticizer, or other excipients used in pharmaceutical coating compositions.

In another embodiment, the solid composition resembles a coating layer that is coated on multiple units made of insoluble material. Examples of insoluble units include beads made of glass, polymers, metals, and mineral salts. Again, the desired effect is primarily rapid disintegration of the coating layer and quick drug dissolution, which is achieved by providing the solid composition in a physical form that has a particularly high surface-to-volume ratio. Typically, the coating composition will, in addition to the drug and the water-soluble low molecular weight excipient, comprise one or more further excipients, such as those mentioned above for coating soluble particles, or any other excipient known to be useful in pharmaceutical coating compositions.

To achieve the desired effects, it may be useful to incorporate more than one water-soluble low molecular weight excipient into the solid composition. For instance, one excipient may be selected for its drug carrier and diluent capability, while another excipient may be selected to adjust the pH. If the final liquid composition needs to be buffered, two excipients that together form a buffer system may be selected.

In one embodiment, the liquid to be used in a separated-compartment nebulizer is an aqueous liquid, which is herein defined as a liquid whose major component is water. The liquid does not necessarily consist of water only; however, in one embodiment it is purified water. In another embodiment, the liquid contains other components or substances, preferably other liquid components, but possibly also dissolved solids. Liquid components other than water which may be useful include propylene glycol, glycerol, and polyethylene glycol. One of the reasons to incorporate a solid compound as a solute is that such a compound is needed or desirable in the final liquid composition, but is incompatible with the solid composition or with a component thereof, such as the active ingredient.

Another requirement for the liquid solvent is that it is sterile. An aqueous liquid would be subject to the risk of considerable microbiological contamination and growth if no measures were taken to ensure sterility. In order to provide a substantially sterile liquid, it is either necessary to incorporate an effective amount of an acceptable antimicrobial agent or preservative, or to sterilize the liquid prior to providing it and to seal it with an air-tight seal. In one embodiment, the liquid is a sterilized liquid free of preservatives and provided in an appropriate air-tight container. However, according to another embodiment in which the nebulizer contains multiple doses of the active compound, the liquid may be supplied in a multiple-dose container, such as a metered-dose dispenser, and may require a preservative to prevent microbial contamination after the first use.

Meter Dose Inhaler (MDI)

A propellant driven inhaler (pMDI) releases a metered dose of medicine upon each actuation. The medicine is formulated as a suspension or solution of a drug substance in a suitable propellant such as a halogenated hydrocarbon. pMDIs are described in, for example, Newman, S. P., Aerosols and the Lung, Clarke et al., eds., pp. 197-224 (Butterworths, London, England, 1984).

As in the case for MDIs and nebulizers, the particle size of the drug substance in a DPI may be optimally chosen. In some embodiments, the particles of active ingredient have diameters of less than 50 microns. In some embodiments, the particles have diameters of less than 10 microns. In some embodiments, the particles have diameters of between 1 micron and 5 microns. In some embodiments, the particles have diameters of less than 1 micron. In one advantageous embodiment, the particles have diameters of between 2 microns and 4 microns.

The propellants for use with the MDIs may be any propellants known in the art. Examples of propellants include chlorofluorocarbons (CFCs) such as dichlorodifluoromethane, trichlorofluorometbane, and dichlorotetrafluoroethane; hydrofluoroalkanes (HFAs); and carbon dioxide. It may be advantageous to use HFAs instead of CFCs due to the environmental concerns associated with the use of CFCs. Examples of medicinal aerosol preparations containing HFAs are presented in U.S. Pat. Nos. 6,585,958; 2,868,691 and 3,014,844, all of which are hereby incorporated by reference in their entirety. In some embodiments, a co-solvent is mixed with the propellant to facilitate dissolution or suspension of the drug substance.

In some embodiments, the propellant and active ingredient are contained in separate containers, such as described in U.S. Pat. No. 4,534,345, which is hereby incorporated by reference in its entirety.

In some embodiments, the MDI used herein is activated by a patient pushing a lever, button, or other actuator. In other embodiments, the release of the aerosol is breath activated such that, after initially arming the unit, the active compound aerosol is released once the patient begins to inhale, such as described in U.S. Pat. Nos. 6,672,304; 5,404,871; 5,347,998; 5,284,133; 5,217,004; 5,119,806; 5,060,643; 4,664,107; 4,648,393; 3,789,843; 3,732,864; 3,636,949; 3,598,294; 3,565,070; 3,456,646; 3,456,645; and 3,456,644, each of which is hereby incorporated by reference in its entirety. Such a system enables more of the active compound to get into the lungs of the patient. Another mechanism to help a patient get adequate dosage with the active ingredient may include a valve mechanism that allows a patient to use more than one breath to inhale the drug, such as described in U.S. Pat. Nos. 4,470,412 and 5,385,140, both of which are hereby incorporated by reference in their entirety.

Additional examples of MDIs known in the art and suitable for use herein include U.S. Pat. Nos. 6,435,177; 6,585,958; 5,642,730; 6,223,746; 4,955,371; 5,404,871; 5,364,838; and 6,523,536, all of which are hereby incorporated by reference in their entirety.

Dry Powder Inhaler (DPI)

There are two major designs of dry powder inhalers. One design is the metering device in which a reservoir for the drug is placed within the device and the patient adds a dose of the drug into the inhalation chamber. The second is a factory-metered device in which each individual dose has been manufactured in a separate container. Both systems depend upon the formulation of drug into small particles of mass median diameters from 1 to 5 μm, and usually involve co-formulation with larger excipient particles (typically 100 μm diameter lactose particles). Drug powder is placed into the inhalation chamber (either by device metering or by breakage of a factory-metered dosage) and the inspiratory flow of the patient accelerates the powder out of the device and into the oral cavity. Non-laminar flow characteristics of the powder path cause the excipient-drug aggregates to decompose, and the mass of the large excipient particles causes their impaction at the back of the throat, while the smaller drug particles are deposited deep in the lungs.

As with liquid nebulization, particle size of the pentamidine or pentamidine analog in the presence or absence of antibiotic aerosol formulation may be optimized. If the particle size is larger than 5 μm MMAD then the particles are deposited in upper airways. If the particle size of the aerosol is smaller the 1 μm then it is delivered into the alveoli and may get transferred into the systemic blood circulation.

By non-limiting example, in dry powder inhalers, the efflux pump inhibitors disclosed herein in the presence or absence of antibiotic dry powder are prepared in dosages from 1-100 mg, preferably from 10-60 mg of dry powder as particles having sizes between 1 and 4 μm.

In some embodiments, a dry powder inhaler (DPI) is used to dispense the EPIs described herein. DPIs contain the drug substance in fine dry particle form. Typically, inhalation by a patient causes the dry particles to form an aerosol cloud that is drawn into the patient's lungs. The fine dry drug particles may be produced by any technique known in the art. Some well-known techniques include use of a jet mill or other comminution equipment, precipitation from saturated or super saturated solutions, spray drying, or supercritical fluid methods. Typical powder formulations include production of spherical pellets or adhesive mixtures. In adhesive mixtures, the drug particles are attached to larger carrier particles, such as lactose monohydrate of size 50 to 100 microns in diameter. The larger carrier particles increase the aerodynamic forces on the carrier/drug agglomerates to improve aerosol formation. Turbulence and/or mechanical devices break the agglomerates into their constituent parts. The smaller drug particles are then drawn into the lungs while the larger carrier particles deposit in the mouth or throat. Some examples of adhesive mixtures are described in U.S. Pat. No. 5,478,578 and PCT Publication Nos. WO 95/11666, WO 87/05213, WO 96/23485, and WO 97/03649, all of which are incorporated by reference in their entirety. Additional excipients may also be included with the drug substance.

There are three common types of DPIs, all of which may be used with the EPIs described herein. In a single-dose DPI, a capsule containing one dose of dry drug substance/excipients is loaded into the inhaler. Upon activation, the capsule is breached, allowing the dry powder to be aerosolized. To dispense additional doses, the old capsule must be removed and an additional capsule loaded. Examples of single-dose DPIs are described in U.S. Pat. Nos. 3,807,400; 3,906,950; 3,991,761; and 4,013,075, all of which are hereby incorporated by reference in their entirety. In a multiple unit dose DPI, a package containing multiple single dose compartments is provided. For example, the package may comprise a blister pack, where each blister compartment contains one dose. Each dose can be dispensed upon breach of a blister compartment. Any of several arrangements of compartments in the package can be used. For example, rotary or strip arrangements are common. Examples of multiple unit does DPIs are described in EPO Patent Application Publication Nos. 0211595A2, 0455463A1, and 0467172A1, all of which are hereby incorporated by reference in their entirety. In a multi-dose DPI, a single reservoir of dry powder is used. Mechanisms are provided that measure out single dose amounts from the reservoir to be aerosolized and inhaled, such as described in U.S. Pat. Nos. 5,829,434; 5,437,270; 2,587,215; 5,113,855; 5,840,279; 4,688,218; 4,667,668; 5,033,463; and 4,805,811 and PCT Publication No. WO 92/09322, all of which are hereby incorporated by reference in their entirety.

In some embodiments, auxiliary energy in addition to or other than a patient's inhalation may be provided to facilitate operation of a DPI. For example, pressurized air may be provided to aid in powder de-agglomeration, such as described in U.S. Pat. Nos. 3,906,950; 5,113,855; 5,388,572; 6,029,662 and PCT Publication Nos. WO 93/12831, WO 90/07351, and WO 99/62495, all of which are hereby incorporated by reference in their entirety. Electrically driven impellers may also be provided, such as described in U.S. Pat. Nos. 3,948,264; 3,971,377; 4,147,166; 6,006,747 and PCT Publication No. WO 98/03217, all of which are hereby incorporated by reference in their entirety. Another mechanism is an electrically powered tapping piston, such as described in PCT Publication No. WO 90/13327, which is hereby incorporated by reference in its entirety. Other DPIs use a vibrator, such as described in U.S. Pat. Nos. 5,694,920 and 6,026,809, both of which are hereby incorporated by reference in their entirety. Finally, a scraper system may be employed, such as described in PCT Publication No. WO 93/24165, which is hereby incorporated by reference in its entirety.

Additional examples of DPIs for use herein are described in U.S. Pat. Nos. 4,811,731; 5,113,855; 5,840,279; 3,507,277; 3,669,113; 3,635,219; 3,991,761; 4,353,365; 4,889,144, 4,907,538; 5,829,434; 6,681,768; 6,561,186; 5,918,594; 6,003,512; 5,775,320; 5,740,794; and 6,626,173, all of which are hereby incorporated by reference in their entirety.

In some embodiments, a spacer or chamber may be used with any of the inhalers described herein to increase the amount of drug substance that gets absorbed by the patient, such as is described in U.S. Pat. Nos. 4,470,412; 4,790,305; 4,926,852; 5,012,803; 5,040,527; 5,024,467; 5,816,240; 5,027,806; and 6,026,807, all of which are hereby incorporated by reference in their entirety. For example, a spacer may delay the time from aerosol production to the time when the aerosol enters a patient's mouth. Such a delay may improve synchronization between the patient's inhalation and the aerosol production. A mask may also be incorporated for infants or other patients that have difficulty using the traditional mouthpiece, such as is described in U.S. Pat. Nos. 4,809,692; 4,832,015; 5,012,804; 5,427,089; 5,645,049; and 5,988,160, all of which are hereby incorporated by reference in their entirety.

Pentamidine formulations for use in inhalers have been described for the treatment of Pneumocystis carinii pneumonia. For example, pentamidine formulations for nebulizer dispensing are described in U.S. Pat. Nos. 5,364,615; 5,366,726; 4,853,416; 5,084,480; 5,262,157; and 5,089,527, which are all hereby incorporated by reference in their entirety. Dry pentamidine formulations for dispensing by an MDI or DPI are described in U.S. Pat. Nos. 5,204,113, and 5,334,374, which are both hereby incorporated by reference in their entirety. These formulations and dispensing methods may be used for the efflux pump inhibiting described herein.

Dry powder inhalers (DPIs), which involve deaggregation and aerosolization of dry powders, normally rely upon a burst of inspired air that is drawn through the unit to deliver a drug dosage. Such devices are described in, for example, U.S. Pat. No. 4,807,814, which is directed to a pneumatic powder ejector having a suction stage and an injection stage; SU 628930 (Abstract), describing a hand-held powder disperser having an axial air flow tube; Fox et al., Powder and Bulk Engineering, pages 33-36 (March 1988), describing a venturi eductor having an axial air inlet tube upstream of a venturi restriction; EP 347 779, describing a hand-held powder disperser having a collapsible expansion chamber, and U.S. Pat. No. 5,785,049, directed to dry powder delivery devices for drugs.

Solution/Dispersion Formulations

In one embodiment, aqueous formulations containing soluble or nanoparticulate drug particles are provided. For aqueous aerosol formulations, the drug may be present at a concentration of about 0.05 mg/mL up to about 600 mg/mL. Such formulations provide effective delivery to appropriate areas of the lung or nasal cavities. In addition, the more concentrated aerosol formulations (i.e., for aqueous aerosol formulations, about 10 mg/mL up to about 600 mg/mL) have the additional advantage of enabling large quantities of drug substance to be delivered to the lung in a very short periods of time. In one embodiment, a formulation is optimized to provide a well tolerated formulation. Accordingly, in one embodiment, efflux pump inhibitors disclosed herein (in the presence or absence of antibiotic) are formulated to have good taste, pH between 5.5-7, osmolarity between 150-550 mOsm/kg, permeant ion concentration between 31-300 mM.

In one embodiment, the solution or diluent used for preparation of aerosol formulations has a pH range from 4.5 to 7.5, preferably between 5.5 and 7.0. This pH range improves tolerability. When the aerosol is either acidic or basic, it can cause bronchospasm and cough. Although the safe range of pH is relative and some patients may tolerate a mildly acidic aerosol, while others will experience bronchospasm. Any aerosol with a pH of less than 4.5 typically induces bronchospasm. Aerosols with a pH between 4.5 and 5.5 will cause bronchospasm occasionally. Any aerosol having pH greater than 7.5 is to be avoided as the body tissues are unable to buffer alkaline aerosols. Aerosol with controlled pH below 4.5 and over 7.5 result in lung irritation accompanied by severe bronchospasm cough and inflammatory reactions. For these reasons as well as for the avoidance of bronchospasm, cough or inflammation in patients, the optimum pH for the aerosol formulation was determined to be between pH 5.5 to pH 7.0. Consequently, in one embodiment, aerosol formulations for use as described herein are adjusted to pH between 4.5 and 7.5 with preferred pH range from about 5.5 to 7.0. Most preferred pH range is from 5.5 to 6.5.

By non-limiting example, compositions may also include a buffer or a pH adjusting agent, typically a salt prepared from an organic acid or base. Representative buffers include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride, or phosphate buffers.

Many patients have increased sensitivity to various chemical agents and have high incidence of bronchospastic, asthmatic or other coughing incidents. Their airways are particularly sensitive to hypotonic or hypertonic and acidic or alkaline conditions and to the presence of any permanent ion, such as chloride. Any imbalance in these conditions or a presence of chloride above certain value leads to bronchospastic or inflammatory events and/or cough which greatly impair treatment with inhalable formulations. Both these conditions prevent efficient delivery of aerosolized drugs into the endobronchial space.

In some embodiments, the osmolarity of aqueous solutions of the efflux pump inhibitors disclosed herein are adjusted by providing excipients. In some cases, a certain amount of chloride or another anion is needed for successful and efficacious delivery of aerosolized aztreonam. However, it has been discovered that such amounts are lower than amounts provided and typically used for aerosols of other compounds.

Bronchospasm or cough reflexes do not respond to the same osmolarity of the diluent for aerosolization. However, they can be sufficiently controlled and/or suppressed when the osmolarity of the diluent is in a certain range. A preferred solution for aerosolization of therapeutic compounds which is safe and tolerated has a total osmolarity between 150 and 550 mOsm/kg with a range of chloride concentration of between 31 mM and 300 mM. This osmolarity controls bronchospasm, the chloride concentration, as a permeant anion, controls cough. Because they are both permeant ions, both bromine or iodine anions may be substituted for chloride. In addition, bicarbonate may substituted for chloride ion.

By non-limiting example, the formulation for aerosol efflux pump inhibitors in the presence or absence of antibiotic may comprise ˜1-100 mg, preferably about ˜2-50 mg pentamidine or pentamidine analog per 1 ml of dilute saline (between 1/10 to ¾ normal saline). Moreover, the compounds (in the presence or absence of antibiotic) may have an osmolarity between 150 and 550 mOsm/kg. Such osmolarity is within a safe range of aerosols suitable for administration to patients suffering from pulmonary bacterial infections.

In one embodiment, solution osmolarity is from about 100 mOsmol/kg to about 600 mOsmol/kg. In various other embodiments, the solution osmolarity is from about 150 mOsmol/kg to about 550 mOsmol/kg; from about 200 mOsmol/kg to about 500 mOsmol/kg; from about 250 mOsmol/kg to about 450 mOsmol/kg; and from about 300 mOsmol/kg to about 400 mOsmol/kg.

In one embodiments, permeant ion concentration is from about 25 mM to about 400 mM. In various other embodiments, permeant ion concentration is from about 50 mM to about 300 mM; from about 50 mM to about 250 mM; from about 75 mM to about 200 mM; and from about 100 mM to about 150 mM.

Solid Particle Formulations

In some embodiments, solid drug nanoparticles are provided for use in generating dry aerosols or for generating nanoparticles in liquid suspension. Powders comprising nanoparticulate drug can be made by spray-drying aqueous dispersions of a nanoparticulate drug and a surface modifier to form a dry powder which consists of aggregated drug nanoparticles. In one embodiment, the aggregates can have a size of about 1 to about 2 microns which is suitable for deep lung delivery. The aggregate particle size can be increased to target alternative delivery sites, such as the upper bronchial region or nasal mucosa by increasing the concentration of drug in the spray-dried dispersion or by increasing the droplet size generated by the spray dryer.

Alternatively, an aqueous dispersion of drug and surface modifier can contain a dissolved diluent such as lactose or mannitol which, when spray dried, forms respirable diluent particles, each of which contains at least one embedded drug nanoparticle and surface modifier. The diluent particles with embedded drug can have a particle size of about 1 to about 2 microns, suitable for deep lung delivery. In addition, the diluent particle size can be increased to target alternate delivery sites, such as the upper bronchial region or nasal mucosa by increasing the concentration of dissolved diluent in the aqueous dispersion prior to spray drying, or by increasing the droplet size generated by the spray dryer.

Spray-dried powders can be used in DPIs or pMDIs, either alone or combined with freeze-dried nanoparticulate powder. In addition, spray-dried powders containing drug nanoparticles can be reconstituted and used in either jet or ultrasonic nebulizers to generate aqueous dispersions having respirable droplet sizes, where each droplet contains at least one drug nanoparticle. Concentrated nanoparticulate dispersions may also be used in these aspects of the invention.

Nanoparticulate drug dispersions can also be freeze-dried to obtain powders suitable for nasal or pulmonary delivery. Such powders may contain aggregated nanoparticulate drug particles having a surface modifier. Such aggregates may have sizes within a respirable range, e.g., about 2 to about 4 microns MMAD. Larger aggregate particle sizes can be obtained for targeting alternate delivery sites, such as the nasal mucosa

Freeze dried powders of the appropriate particle size can also be obtained by freeze drying aqueous dispersions of drug and surface modifier, which additionally contain a dissolved diluent such as lactose or mannitol. In these instances the freeze dried powders consist of respirable particles of diluent, each of which contains at least one embedded drug nanoparticle.

Freeze-dried powders can be used in DPIs or pMDIs, either alone or combined with spray-dried nanoparticulate powder. In addition, freeze-dried powders containing drug nanoparticles can be reconstituted and used in either jet or ultrasonic nebulizers to generate aqueous dispersions that have respirable droplet sizes, where each droplet contains at least one drug nanoparticle.

One embodiment of the invention is directed to a process and composition for propellant-based systems comprising nanoparticulate drug particles and a surface modifier. Such formulations may be prepared by wet milling the coarse drug substance and surface modifier in liquid propellant, either at ambient pressure or under high pressure conditions. Alternatively, dry powders containing drug nanoparticles may be prepared by spray-drying or freeze-drying aqueous dispersions of drug nanoparticles and the resultant powders dispersed into suitable propellants for use in conventional pMDIs. Such nanoparticulate pMDI formulations can be used for either nasal or pulmonary delivery. For pulmonary administration, such formulations afford increased delivery to the deep lung regions because of the small (e.g., about 1 to about 2 microns MMAD) particle sizes available from these methods. Concentrated aerosol formulations can also be employed in pMDIs.

Another embodiment is directed to dry powders which contain nanoparticulate compositions for pulmonary or nasal delivery. The powders may consist of respirable aggregates of nanoparticulate drug particles, or of respirable particles of a diluent which contains at least one embedded drug nanoparticle. Powders containing nanoparticulate drug particles can be prepared from aqueous dispersions of nanoparticles by removing the water via spray-drying or lyophilization (freeze drying). Spray-drying is less time consuming and less expensive than freeze-drying, and therefore more cost-effective. However, certain drugs, such as biologicals benefit from lyophilization rather than spray-drying in making dry powder formulations.

Conventional micronized drug particles used in dry powder aerosol delivery having partcticle diameters of 2-3 microns MMAD are often difficult to meter and disperse in small quantities because of the electrostatic cohesive forces inherent in such powders. These difficulties can lead to loss of drug substance to the delivery device as well as incomplete powder dispersion and sub-optimal delivery to the lung. Many drug compounds, particularly proteins and peptides, are intended for deep lung delivery and systemic absorption. Since the average particle sizes of conventionally prepared dry powders are usually in the range of 2-3 microns MMAD, the fraction of material which actually reaches the alveolar region may be quite small. Thus, delivery of micronized dry powders to the lung, especially the alveolar region, is generally very inefficient because of the properties of the powders themselves.

The dry powder aerosols which contain nanoparticulate drugs can be made smaller than comparable micronized drug substance and, therefore, are appropriate for efficient delivery to the deep lung. Moreover, aggregates of nanoparticulate drugs are spherical in geometry and have good flow properties, thereby aiding in dose metering and deposition of the administered composition in the lung or nasal cavities.

Dry nanoparticulate compositions can be used in both DPIs and pMDIs. As used herein, “dry” refers to a composition having less than about 5% water.

In one embodiment, compositions are provided containing nanoparticles which have an effective average particle size of less than about 1000 nm, more preferably less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 100 nm, or less than about 50 nm, as measured by light-scattering methods. By “an effective average particle size of less than about 1000 nm” it is meant that at least 50% of the drug particles have a weight average particle size of less than about 1000 nm when measured by light scattering techniques. Preferably, at least 70% of the drug particles have an average particle size of less than about 1000 nm, more preferably at least 90% of the drug particles have an average particle size of less than about 1000 nm, and even more preferably at least about 95% of the particles have a weight average particle size of less than about 1000 nm.

For aqueous aerosol formulations, the nanoparticulate agent may be present at a concentration of about 0.05 mg/mL up to about 600 mg/mL. For dry powder aerosol formulations, the nanoparticulate agent may be present at a concentration of about 0.05 mg/g up to about 990 mg/g, depending on the desired drug dosage. Concentrated nanoparticulate aerosols, defined as containing a nanoparticulate drug at a concentration of about 10 mg/mL up to about 600 mg/mL for aqueous aerosol formulations, and about 10 mg/g up to about 990 mg/g for dry powder aerosol formulations, are specifically provided. Such formulations provide effective delivery to appropriate areas of the lung or nasal cavities in short administration times, ie., less than about 15 seconds as compared to administration times of up to 4 to 20 minutes as found in conventional pulmonary nebulizer therapies.

Nanoparticulate drug compositions for aerosol administration can be made by, for example, (1) nebulizing a dispersion of a nanoparticulate drug, obtained by either grinding or precipitation; (2) aerosolizing a dry powder of aggregates of nanoparticulate drug and surface modifier (the aerosolized composition may additionally contain a diluent); or (3) aerosolizing a suspension of nanoparticulate drug or drug aggregates in a non-aqueous propellant. The aggregates of nanoparticulate drug and surface modifier, which may additionally contain a diluent, can be made in a non-pressurized or a pressurized non-aqueous system. Concentrated aerosol formulations may also be made via such methods.

Milling of aqueous drug to obtain nanoparticulate drug may be performed by dispersing drug particles in a liquid dispersion medium and applying mechanical means in the presence of grinding media to reduce the particle size of the drug to the desired effective average particle size. The particles can be reduced in size in the presence of one or more surface modifiers. Alternatively, the particles can be contacted with one or more surface modifiers after attrition. Other compounds, such as a diluent, can be added to the drug/surface modifier composition during the size reduction process. Dispersions can be manufactured continuously or in a batch mode.

Another method of forming nanoparticle dispersion is by microprecipitation. This is a method of preparing stable dispersions of drugs in the presence of one or more surface modifiers and one or more colloid stability enhancing surface active agents free of any trace toxic solvents or solubilized heavy metal impurities. Such a method comprises, for example, (1) dissolving the drug in a suitable solvent with mixing; (2) adding the formulation from step (1) with mixing to a solution comprising at least one surface modifier to form a clear solution; and (3) precipitating the formulation from step (2) with mixing using an appropriate nonsolvent. The method can be followed by removal of any formed salt, if present, by dialysis or diafiltration and concentration of the dispersion by conventional means. The resultant nanoparticulate drug dispersion can be utilized in liquid nebulizers or processed to form a dry powder for use in a DPI or pMDI.

In a non-aqueous, non-pressurized milling system, a non-aqueous liquid having a vapor pressure of about 1 atm or less at room temperature and in which the drug substance is essentially insoluble may be used as a wet milling medium to make a nanoparticulate drug composition. In such a process, a slurry of drug and surface modifier may be milled in the non-aqueous medium to generate nanoparticulate drug particles. Examples of suitable non-aqueous media include ethanol, trichloromonofluoromethane, (CFC-11), and dichlorotetafluoroethane (CFC-114). An advantage of using CFC-11 is that it can be handled at only marginally cool room temperatures, whereas CFC-114 requires more controlled conditions to avoid evaporation. Upon completion of milling the liquid medium may be removed and recovered under vacuum or heating, resulting in a dry nanoparticulate composition. The dry composition may then be filled into a suitable container and charged with a final propellant. Exemplary final product propellants, which ideally do not contain chlorinated hydrocarbons, include HFA-134a (tetrafluoroethane) and HFA-227 (heptafluoropropane). While non-chlorinated propellants may be preferred for environmental reasons, chlorinated propellants may also be used in this aspect of the invention.

In a non-aqueous, pressurized milling system, a non-aqueous liquid medium having a vapor pressure significantly greater than 1 atm at room temperature may be used in the milling process to make nanoparticulate drug compositions. If the milling medium is a suitable halogenated hydrocarbon propellant, the resultant dispersion may be filled directly into a suitable pMDI container. Alternately, the milling medium can be removed and recovered under vacuum or heating to yield a dry nanoparticulate composition. This composition can then be filled into an appropriate container and charged with a suitable propellant for use in a pMDI.

Spray drying is a process used to obtain a powder containing nanoparticulate drug particles following particle size reduction of the drug in a liquid medium. In general, spray-drying may be used when the liquid medium has a vapor pressure of less than about 1 atm at room temperature. A spray-dryer is a device which allows for liquid evaporation and drug powder collection. A liquid sample, either a solution or suspension, is fed into a spray nozzle. The nozzle generates droplets of the sample within a range of about 20 to about 100 μm in diameter which are then transported by a carrier gas into a drying chamber. The carrier gas temperature is typically between about 80 and about 200° C. The droplets are subjected to rapid liquid evaporation, leaving behind dry particles which are collected in a special reservoir beneath a cyclone apparatus.

If the liquid sample consists of an aqueous dispersion of nanoparticles and surface modifier, the collected product will consist of spherical aggregates of the nanoparticulate drug particles. If the liquid sample consists of an aqueous dispersion of nanoparticles in which an inert diluent material was dissolved (such as lactose or mannitol), the collected product will consist of diluent (e.g., lactose or mannitol) particles which contain embedded nanoparticulate drug particles. The final size of the collected product can be controlled and depends on the concentration of nanoparticulate drug and/or diluent in the liquid sample, as well as the droplet size produced by the spray-dryer nozzle. Collected products may be used in conventional DPIs for pulmonary or nasal delivery, dispersed in propellants for use in pMDIs, or the particles may be reconstituted in water for use in nebulizers.

In some instances it may be desirable to add an inert carrier to the spray-dried material to improve the metering properties of the final product. This may especially be the case when the spray dried powder is very small (less than about 5 μm) or when the intended dose is extremely small, whereby dose metering becomes difficult. In general, such carrier particles (also known as bulking agents) are too large to be delivered to the lung and simply impact the mouth and throat and are swallowed. Such carriers typically consist of sugars such as lactose, mannitol, or trehalose. Other inert materials, including polysaccharides and cellulosics, may also be useful as carriers.

Spray-dried powders containing nanoparticulate drug particles may used in conventional DPIs, dispersed in propellants for use in pMDIs, or reconstituted in a liquid medium for use with nebulizers.

For compounds that are denatured or destabilized by heat, such as compounds having a low melting point (i.e., about 70 to about 150° C.), or for example, biologics, sublimation is preferred over evaporation to obtain a dry powder nanoparticulate drug composition. This is because sublimation avoids the high process temperatures associated with spray-drying. In addition, sublimation, also known as freeze-drying or lyophilization, can increase the shelf stability of drug compounds, particularly for biological products. Freeze-dried particles can also be reconstituted and used in nebulizers. Aggregates of freeze-dried nanoparticulate drug particles can be blended with either dry powder intermediates or used alone in DPIs and pMDIs for either nasal or pulmonary delivery.

Sublimation involves freezing the product and subjecting the sample to strong vacuum conditions. This allows for the formed ice to be transformed directly from a solid state to a vapor state. Such a process is highly efficient and, therefore, provides greater yields than spray-drying. The resultant freeze-dried product contains drug and modifier(s). The drug is typically present in an aggregated state and can be used for inhalation alone (either pulmonary or nasal), in conjunction with diluent materials (lactose, mannitol, etc.), in DPIs or pMDIs, or reconstituted for use in a nebulizer.

Liposomal Compositions

In some embodiments, efflux pump inhibitors disclosed herein may be formulated into liposome particles, which can then be aerosolized for inhaled delivery. Lipids which are useful in the present invention can be any of a variety of lipids including both neutral lipids and charged lipids. Carrier systems having desirable properties can be prepared using appropriate combinations of lipids, targeting groups and circulation enhancers. Additionally, the compositions provided herein can be in the form of liposomes or lipid particles, preferably lipid particles. As used herein, the term “lipid particle” refers to a lipid bilayer carrier which “coats” a nucleic acid and has little or no aqueous interior. More particularly, the term is used to describe a self-assembling lipid bilayer carrier in which a portion of the interior layer comprises cationic lipids which form ionic bonds or ion-pairs with negative charges on the nucleic acid (e.g., a plasmid phosphodiester backbone). The interior layer can also comprise neutral or fusogenic lipids and, in some embodiments, negatively charged lipids. The outer layer of the particle will typically comprise mixtures of lipids oriented in a tail-to-tail fashion (as in liposomes) with the hydrophobic tails of the interior layer. The polar head groups present on the lipids of the outer layer will form the external surface of the particle.

Liposomal bioactive agents can be designed to have a sustained therapeutic effect or lower toxicity allowing less frequent administration and an enhanced therapeutic index. Liposomes are composed of bilayers that entrap the desired pharmaceutical. These can be configured as multilamellar vesicles of concentric bilayers with the pharmaceutical trapped within either the lipid of the different layers or the aqueous space between the layers.

By non-limiting example, lipids used in the compositions may be synthetic, semi-synthetic or naturally-occurring lipids, including phospholipids, tocopherols, steroids, fatty acids, glycoproteins such as albumin, negatively-charged lipids and cationic lipids. Phosholipids include egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and egg phosphatidic acid (EPA); the soya counterparts, soy phosphatidylcholine (SPC); SPG, SPS, SPI, SPE, and SPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC), other phospholipids made up of ester linkages of fatty acids in the 2 and 3 of glycerol positions containing chains of 12 to 26 carbon atoms and different head groups in the 1 position of glycerol that include choline, glycerol, inositol, serine, ethanolamine, as well as the corresponding phosphatidic acids. The chains on these fatty acids can be saturated or unsaturated, and the phospholipid can be made up of fatty acids of different chain lengths and different degrees of unsaturation. In particular, the compositions of the formulations can include dipalmitoylphosphatidylcholine (DPPC), a major constituent of naturally-occurring lung surfactant as well as dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). Other examples include dimyristoylphosphatidycholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE) and mixed phospholipids like palmitoylstearoylphosphatidylcholine (PSPC) and palmitoylstearoylphosphatidylglycerol (PSPG), and single acylated phospholipids like mono-oleoyl-phosphatidylethanolamine (MOPE).

In a preferred embodiment, PEG-modified lipids are incorporated into the compositions of the present invention as the aggregation-preventing agent. The use of a PEG-modified lipid positions bulky PEG groups on the surface of the liposome or lipid carrier and prevents binding of DNA to the outside of the carrier (thereby inhibiting cross-linking and aggregation of the lipid carrier). The use of a PEG-ceramide is often preferred and has the additional advantages of stabilizing membrane bilayers and lengthening circulation lifetimes. Additionally, PEG-ceramides can be prepared with different lipid tail lengths to control the lifetime of the PEG-ceramide in the lipid bilayer. In this manner, “programmable” release can be accomplished which results in the control of lipid carrier fusion. For example, PEG-ceramides having C₂₀-acyl groups attached to the ceramide moiety will diffuse out of a lipid bilayer carrier with a half-life of 22 hours. PEG-ceramides having C₁₄- and C₈-acyl groups will diffuse out of the same carrier with half-lives of 10 minutes and less than 1 minute, respectively. As a result, selection of lipid tail length provides a composition in which the bilayer becomes destabilized (and thus fusogenic) at a known rate. Though less preferred, other PEG-lipids or lipid-polyoxyethylene conjugates are useful in the present compositions. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-modified diacylglycerols and dialkylglycerols, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-ceramide conjugates (e.g., PEG-Cer-C₈, PEG-Cer-C₁₄ or PEG-Cer-C₂₀) which are described in U.S. Pat. No. 5,820,873, incorporated herein by reference.

The compositions of the present invention can be prepared to provide liposome compositions which are about 50 nm to about 400 nm in diameter. One of skill in the art will understand that the size of the compositions can be larger or smaller depending upon the volume which is encapsulated. Thus, for larger volumes, the size distribution will typically be from about 80 nm to about 300 nm.

Surface Modifiers

Efflux pump inhibitors disclosed herein, in the presence or absence of antibiotic, may be prepared in a pharmaceutical composition with suitable surface modifiers which may be selected from known organic and inorganic pharmaceutical excipients. Such excipients include low molecular weight oligomers, polymers, surfactants and natural products. Preferred surface modifiers include nonionic and ionic surfactants. Two or more surface modifiers can be used in combination.

Representative examples of surface modifiers include cetyl pyridinium chloride, gelatin, casein, lecithin (phosphatides), dextran, glycerol, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers (e.g., macrogol ethers such as cetomacrogol 1000), polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters (e.g., the commercially available Tweens® such as e.g., Tween 20® and Tween 80® (ICI Specialty Chemicals)); polyethylene glycols (e.g., Carbowaxs 3350® and 1450®, and Carbopol 934® (Union Carbide)), dodecyl trimethyl ammonium bromide, polyoxyethylenestearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, hydroxypropyl cellulose (HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), 4-(1,1,3,3-tetaamethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol, superione, and triton), poloxamers (e.g., Pluronics F68® and F108®, which are block copolymers of ethylene oxide and propylene oxide); poloxamnines (e.g., Tetronic 908®, also known as Poloxamine 908®, which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Wyandotte Corporation, Parsippany, N.J.)); a charged phospholipid such as dimyristoyl phophatidyl glycerol, dioctylsulfosuccinate (DOSS); Tetronic 1508® (T-1508) (BASF Wyandotte Corporation), dialkylesters of sodium sulfosuccinic acid (e.g., Aerosol OT®, which is a dioctyl ester of sodium sulfosuccinic acid (American Cyanamid)); Duponol P®, which is a sodium lauryl sulfate (DuPont); Tritons X-200, which is an alkyl aryl polyether sulfonate (Rohm and Haas); Crodestas F-110®, which is a mixture of sucrose stearate and sucrose distearate (Croda Inc.); p-isononylphenoxypoly-(glycidol), also known as Olin-log® or Surfactant 10-G® (Olin Chemicals, Stamford, Conn.); Crodestas SL-40® (Croda, Inc.); and SA9OHCO, which is C.sub.18 H.sub.37 CH.sub.2 (CON(CH.sub.3) -CH.sub.2 (CHOH).sub.4 (CH.sub.2 OH).sub.2 (Eastman Kodak Co.); decanoyl-N-methylglucamide; n-decyl .beta. -D-glucopyranoside; n-decyl .beta.-D-maltopyranoside; n-dodecyl .beta.-D-glucopyranoside; n-dodecyl .beta.-D-maltoside; heptanoyl-N-methylglucamide; n-heptyl-.beta.-D-glucopyranoside; n-heptyl .beta.-D-thioglucoside; n-hexyl .beta.-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noyl .beta.-D-glucopyranoside; octanoyl-N-methylglucarmide; n-octyl-.beta.-D-glucopyranoside; octyl .beta.-D-thioglucopyranoside; and the like. Tyloxapol is a particularly preferred surface modifier for the pulmonary or intranasal delivery of steroids, even more so for nebulization therapies.

Most of these surface modifiers are known pharmaceutical excipients and are described in detail in the Handbook of Pharmaceutical Excipients, published jointly by the American Pharmaceutical Association and The Pharmaceutical Society of Great Britain (The Pharmaceutical Press, 1986), specifically incorporated by reference. The surface modifiers are commercially available and/or can be prepared by techniques known in the art. The relative amount of drug and surface modifier can vary widely and the optimal amount of the surface modifier can depend upon, for example, the particular drug and surface modifier selected, the critical micelle concentration of the surface modifier if it forms micelles, the hydrophilic-lipophilic-balance (HLB) of the surface modifier, the melting point of the surface modifier, the water solubility of the surface modifier and/or drug, the surface tension of water solutions of the surface modifier, etc.

In the present invention, the optimal ratio of drug to surface modifier is ˜0.1% to ˜99.9% efflux pump inhibitor in the presence or absence of antibiotic, more preferably about 10% to about 90%.

Dispersion-Enhancing Peptides

Compositions may include one or more di- or tripeptides containing two or more leucine residues. By further non-limiting example, U.S. Pat. No. 6,835,372 disclosing dispersion-enhancing peptides, is hereby incorporated by reference in its entirety. This patent describes the discovery that di-leucyl-containing dipeptides (e.g., dileucine) and tripeptides are superior in their ability to increase the dispersibility of powdered composition.

In another embodiment, highly dispersible particles including an amino acid are administered. Hydrophobic amino acids are preferred. Suitable amino acids include naturally occurring and non-naturally occurring hydrophobic amino acids. Some naturally occurring hydrophobic amino acids, including but not limited to, non-naturally occurring amino acids include, for example, beta-amino acids. Both D, L and racemic configurations of hydrophobic amino acids can be employed. Suitable hydrophobic amino acids can also include amino acid analogs. As used herein, an amino acid analog includes the D or L configuration of an amino acid having the following formula: —NH—CHR—CO—, wherein R is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group and wherein R does not correspond to the side chain of a naturally-occurring amino acid. As used herein, aliphatic groups include straight chained, branched or cyclic C1-C8 hydrocarbons which are completely saturated, which contain one or two heteroatoms such as nitrogen, oxygen or sulfur and/or which contain one or more units of desaturation. Aromatic groups include carbocyclic aromatic groups such as phenyl and naphthyl and heterocyclic aromatic groups such as imidazolyl, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl and acridintyl.

Suitable substituents on an aliphatic, aromatic or benzyl group include —OH, halogen (—Br,—Cl,—I and —F)—O(aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group),—CN, —NO.sub.2, —COOH, —NH.sub.2, —NH(aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), —N(aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group).sub.2, —COO(aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), —CONH.sub.2, —CONH(aliphatic, substituted aliphatic group, benzyl, substituted benzyl, aryl or substituted aryl group)), —SH,—S(aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or substituted aromatic group) and —NH—C(.dbd.NH)—NH.sub.2. A substituted benzylic or aromatic group can also have an aliphatic or substituted aliphatic group as a substituent. A substituted aliphatic group can also have a benzyl, substituted benzyl, aryl or substituted aryl group as a substituent. A substituted aliphatic, substituted aromatic or substituted benzyl group can have one or more substituents. Modifying an amino acid substituent can increase, for example, the lypophilicity or hydrophobicity of natural amino acids which are hydrophilic.

A number of the suitable amino acids, amino acids analogs and salts thereof can be obtained commercially. Others can be synthesized by methods known in the art.

Hydrophobicity is generally defined with respect to the partition of an amino acid between a nonpolar solvent and water. Hydrophobic amino acids are those acids which show a preference for the nonpolar solvent. Relative hydrophobicity of amino acids can be expressed on a hydrophobicity scale on which glycine has the value 0.5. On such a scale, amino acids which have a preference for water have values below 0.5 and those that have a preference for nonpolar solvents have a value above 0.5. As used herein, the term hydrophobic amino acid refers to an amino acid that, on the hydrophobicity scale, has a value greater or equal to 0.5, in other words, has a tendency to partition in the nonpolar acid which is at least equal to that of glycine.

Examples of amino acids which can be employed include, but are not limited to: glycine, proline, alanine, cysteine, methionine, valine, leucine, tyosine, isoleucine, phenylalanine, tryptophan. Preferred hydrophobic amino acids include leucine, isoleucine, alanine, valine, phenylalanine and glycine. Combinations of hydrophobic amino acids can also be employed. Furthermore, combinations of hydrophobic and hydrophilic (preferentially partitioning in water) amino acids, where the overall combination is hydrophobic, can also be employed.

The amino acid can be present in the particles of the invention in an amount of at least 10 weight %. Preferably, the amino acid can be present in the particles in an amount ranging from about 20 to about 80 weight %. The salt of a hydrophobic amino acid can be present in the particles of the invention in an amount of at least 10 weight percent. Preferably, the amino acid salt is present in the particles in an amount ranging from about 20 to about 80 weight %. In preferred embodiments the particles have a tap density of less than about 0.4 g/cm.sup.3.

Methods of forming and delivering particles which include an amino acid are described in U.S. patent application Ser. No 09/382,959, filed on Aug. 25, 1999, entitled Use of Simple Amino Acids to Form Porous Particles During Spray Drying, the teachings of which are incorporated herein by reference in their entirety.

Proteins/Amino Acids

Protein excipients may include albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like. Suitable amino acids (outside of the dileucyl-peptides of the invention), which may also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, tyrosine, tryptophan, and the like. Preferred are amino acids and polypeptides that function as dispersing agents. Amino acids falling into this category include hydrophobic amino acids such as leucine, valine, isoleucine, tryptophan, alanine, methionine, phenylalanine, tyrosine, histidine, and proline. Dispersibility-enhancing peptide excipients include dimers, trimers, tetramers, and pentamers comprising one or more hydrophobic amino acid components such as those described above.

Carbohydrates

By non-limiting example, carbohydrate excipients may include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), pyranosyl sorbitol, myoinositol, isomalt, trehalose and the like.

Polymers

By non-limiting example, compositions may also include polymeric excipients/additives, e.g., polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethylstarch, dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin and sulfobutylether-.beta.-cyclodextrin), polyethylene glycols, and pectin may also be used.

Highly dispersible particles administered comprise a bioactive agent and a biocompatible, and preferably biodegradable polymer, copolymer, or blend. The polymers may be tailored to optimize different characteristics of the particle including: i) interactions between the agent to be delivered and the polymer to provide stabilization of the agent and retention of activity upon delivery; ii) rate of polymer degradation and, thereby, rate of drug release profiles; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity.

Surface eroding polymers such as polyanhydrides may be used to form the particles. For example, polyanhydrides such as poly[(p-carboxyphenoxy)hexane anhydride] (PCPH) may be used. Biodegradable polyanhydrides are described in U.S. Pat. No. 4,857,311. Bulk eroding polymers such as those based on polyesters including poly(hydroxy acids) also can be used. For example, polyglycolic acid (PGA), polylactic acid (PLA), or copolymers thereof may be used to form the particles. The polyester may also have a charged or functionalizable group, such as an amino acid. In a preferred embodiment, particles with controlled release properties can be formed of poly(D,L-lactic acid) and/or poly(DL-lactic-co-glycolic acid) (“PLGA”) which incorporate a surfactant such as dipalmitoyl phosphatidylcholine (DPPC).

Other polymers include polyamides, polycarbonates, polyalkylenes such as polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses and other polysaccharides, and peptides or proteins, or copolymers or blends thereof. Polymers may be selected with or modified to have the appropriate stability and degradation rates in vivo for different controlled drug delivery applications.

Highly dispersible particles can be formed from functionalized polyester graft copolymers, as described in Hrkach et al., Macromolecules, 28: 4736-4739, (1995); and Hrkach et al., “Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class of Functional Biodegradable Biomaterials” in Hydrogels and Biodegradable Polymers for Bioapplications, ACS Symposium Series No. 627, Raphael M. Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp. 93-101, 1996.

In a preferred embodiment of the invention, highly dispersible particles including a bioactive agent and a phospholipid are administered. Examples of suitable phospholipids include, among others, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinositols and combinations thereof. Specific examples of phospholipids include but are not limited to phosphatidylcholines dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidyicholine (DSPC), dipalmitoyl phosphatidyl glycerol (DPPG) or any combination thereof. Other phospholipids are known to those skilled in the art. In a preferred embodiment, the phospholipids are endogenous to the lung.

The phospholipid, can be present in the particles in an amount ranging from about 0 to about 90 weight %. More commonly it can be present in the particles in an amount ranging from about 10 to about 60 weight %.

In another embodiment of the invention, the phospholipids or combinations thereof are selected to impart controlled release properties to the highly dispersible particles. The phase transition temperature of a specific phospholipid can be below, around or above the physiological body temperature of a patient. Preferred phase transition temperatures range from 30.degree. C. to 50.degree. C., (e.g., within .+−.10 degrees of the normal body temperature of patient). By selecting phospholipids or combinations of phospholipids according to their phase transition temperature, the particles can be tailored to have controlled release properties. For example, by administering particles which include a phospholipid or combination of phospholipids which have a phase transition temperature higher than the patient's body temperature, the release of dopamine precursor, agonist or any combination of precursors and/or agonists can be slowed down. On the other hand, rapid release can be obtained by including in the particles phospholipids having lower transition temperatures.

Flavor, Other

By non-limiting example, compositions may further include flavoring agents, taste-masking agents, inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”), sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines), fatty acids and fatty esters, steroids (e.g., cholesterol), and chelating agents (e.g., EDTA, zinc and other such suitable cations). Other pharmaceutical excipients and/or additives suitable for use in the compositions according to the invention are listed in “Remington: The Science & Practice of Pharmacy”, 19.sup.th ed., Williams & Williams, (1995), and in the “Physician's Desk Reference”, 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998).

EXAMPLES

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are incorporated by reference in their entirety.

Example 1 Potentiation of Levofloxacin by Pentamidine

Initial identification of pentamidine as an efflux pump inhibitor was performed by assessing its antibiotic potentiation activity.

Potentiation effect was observed by the reduction of the minimum inhibitory concentration of levofloxacin in the presence of pentamidine. The activity of pentamidine in combination with levofloxacin was assessed by the checkerboard assay (Antimicrobial Combinations. In Antibiotics in Laboratory Medicine, Ed. Victor Lorian, M.D., Fourth edition, 1996, pp 333-338, which is incorporated herein by reference in its entirety) using broth microdilution method performed as recommended by the NCCLS (National Committee for Clinical Laboratory Standards (NCCLS). 1997. Methods for Dilution of Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Fourth Edition; Approved Standard. NCCLS Document M7-A4, Vol 17 No. 2, which is incorporated herein by reference in its entirety). In this assay, multiple dilutions of two drugs, namely pentamidine and levofloxacin, were tested, alone and in combination, at concentrations equal to, above and below their respective minimal inhibitory concentrations (MICs). Pentamidine is readily soluble in water and stock solution was prepared at a final concentration of 10 mg/ml. Stock solutions were further diluted, according to the needs of a particular assay, in Mueller Hinton Broth (MHB). Stock solution was stored at −80° C.

The checkerboard assay was performed in microtiter plates. Levofloxacin was diluted in the x axis, each column containing a single concentration of levofloxacin. Pentamidine was diluted in the y axis, each row containing an equal concentration of pentamidine. The result of these manipulations is that each well of the microtiter plate contains a unique combination of concentrations of the two agents. The assay was performed in MHB with a final bacterial inoculum of 5×10⁵ CFU/ml (from an early-log phase culture). Microtiter plates were incubated during 20 h at 35° C. and were read using a microtiterplate reader (Molecular Devices) at 650 nm as well as visual observation using a microtiter plate reading mirror. The MIC was defined as the lowest concentration of antibiotics, within the combination, at which the visible growth of the organism was completely inhibited.

Levofloxacin potentiation was studied in Pseudomonas aeruginosa. The test organisms used was PAM1020, which is a wild type strain of P. aeruginosa expressing the basal level of MexAB-OprM, as well as PAM1723, PAM1738, and PAM1753, overexpressing the MexAB-OprM, MexCD-OprJ, and MexEF-OprN pump, respectively. In addition levofloxacin potentiation by pentamidine was tested for the strain PAM 1626, which lacks all the three efflux pumps mentioned above. Pentamidine was tested at the maximum concentration of 80 μg/ml. At this concentration, pentamidine demonstrated inhibition of growth of the wild type, however at 20 μg/ml it decreased the levofoxacin MIC in this strain 8-fold. Pentamidine MICs against PAM1723 and PAM1738 were higher than 80μg/ml, however, at 20 μg/ml pentamidine decreased MICs in these strains 8- and 4-fold respectively. Almost no decrease in levofloxacin MIC was seen for PAM1626 lacking efflux pumps. These experiments demonstrated that pentamidine is capable of potentiating levofloxacin activity against strains of P. aeruginosa over-producing efflux pumps but not against the strain which lacks efflux pumps. This result strongly indicates that inhibition of efflux activity is a mechanism of levofloxacin potentiation (Table 1).

Importantly, the MIC of pentamidine against PAM1626 lacking efflux pumps was 10 μg/ml, which is lower than for the strains expressing efflux pumps. This indicates that pentamidine itself is a substrate of these pumps and therefore provides evidence of pentamidine directly interacting with pump proteins. One possible explanation for its pump inhibitory activity in the presence of other substrates such as levofloxacin, is its higher binding affinity to the substrate binding site.

TABLE 1 Potentiation of levofloxacin by pentamidine against the strains of P. aeruginosa expressing or lacking various efflux pumps. Pump which Levofloxacin MIC contributes to MIC* (μg/ml) in the presence of pentamidine (μg/ml) Strain Relevant genotype resistance (μg/ml) 0 1.25 2.5 5 10 20 40 80 PAM1020 wt MexAB-OprM 80 0.25 0.25 0.25 0.125 0.06 0.03 0.015 NG PAM1723 nalB ΔmexCD-oprJ MexAB-OprM >80 2 2 1 1 0.5 0.25 0.06 0.007 ΔmexEF-OprN PAM1738 nfxB ΔmexCD-oprJ MexCD-OprJ >80 0.5 0.5 0.25 0.25 0.25 0.125 0.03 0.015 ΔmexAB-OprM PAM1753 nfxC ΔmexAB-oprM MexEF-OprN 40 4 2 2 2 1 0.25 NG NG ΔmexCD-OprJ PAM1626 ΔmexAB-OprM none 10 0.015 0.007 0.007 0.007 NG NG NG NG ΔmexCD-oprJ ΔmexEF- OprN *MIC for the efflux pump inhinbitor (pentamidine)

Example 2 Potentiation of Efflux Pump Substrates by Pentamidine

While not being bound by any particular theory, it is proposed that pentamidine potentiates antibiotics which are substrates for efflux pumps inhibited by pentamidine. To test this, MICs to several antibiotics for the strain PAM1723 of P. aeruginosa were measured with or without a fixed concentration of pentamidine (40 μg/ml). Pentamidine decreased the MICs of several substrates of MexAB-OprM including levofloxacin (16-fold), ciprofloxacin (8-fold), azithromycin (64-fold), erythromycin (4-fold), and chloramphenicol (16-fold) (Table 2). In contrast, almost no effect of pentamidine was seen for tobramycin, which is not a substrate of this pump (see PAM1723 vs. PAM1154 in Table 2).

TABLE 2 Impact of pentamidine on susceptibility to multiple antibiotics. Impact of EPIs on susceptibility of PAM1723 (MexAB-OprM overexpressed) PAM1154 to various antibiotics (MexAB-OprM Pentamidine deleted) Antibiotic No Pentamidine (40 μg/ml) No Pentamidine Levo 1 0.06 0.015 Cipro 0.25 0.03 0.008 Azithro 64 1 0.5 Erm 256 64 8 Cm 128 8 1 Tb 0.06 0.03 0.03

Example 3 Accumulation Assays

Further proof of efflux pump inhibitory activity of pentamidine was obtained in accumulation assays. Leucine-β-naphthylamide (Leu-Nap) is a substrate of the Mex pumps from P. aeruginosa. Leu-Nap, which is not fluorescent in solution, is cleaved enzymatically inside the cells to produce highly fluorescent β-naphthylamine. The more Leu-Nap enters cells, the more fluorescence is produced. The rate of production of β-naphthylamine (recorded as an increase in fluorescence) is limited by the rate of entry of Leu-Nap into the cell. To assess the uptake of Leu-Nap, cultures of P. aeruginosa were grown to OD₆₀₀ ˜1, washed and re-suspended in buffer at pH 7.0 containing K₂HPO₄ 50 mM, MgSO₄ 1 mM, and Glucose 0.4% (Buffer A). Assays were performed in 96-well flat bottom black plates (Applied Scientific or Costar) in a final volume of 200 μl and were initiated by addition of Leu-Nap to suspensions of intact cells to a final concentration of 100-20 μg/ml. Fluorescence was measured on a fMAX spectrofluorometer (Molecular Devices) using excitation of 320 nm and emission of 460 nm. To measure effects of pentamidine on the rate of Leu-Nap uptake, cells were pre-incubated with different concentrations of pentamidine compounds prior to Leu-Nap addition.

The uptake of Leu-Nap (100 μg/ml) by PAM1723 (FIG. 1A) or PAM1626 (FIG. 1B) cells was studied in the presence of various concentrations (0 μg/ml to 80 μg/ml) of pentamidine. When no pentamidine is added, the rate of cleavage of Leu-Nap was much higher in PAM1626 (FIG. 1B) than in PAM1723 (FIG. 1A—over expressing efflux pump) indicating the Leu-Nap is indeed a substrate of the pump. Addition of pentamidine to PAM1723 cells increases Leu-Nap uptake in the dose-dependent manner (FIG. 1A).

No such increase was seen in the case of PAM1626 cells (lacking the efflux pump). In fact, addition of pentamidine caused a decrease of fluorescence in this latter strain, most probably due to quenching of naphthylamine fluorescence by pentamidine. At a pentamidine concentration of 40 μg/ml (squares in FIGS. 1A and 1B) the rate of uptake in both strains was very similar, indicating that at this concentration pentamidine completely inhibits efflux of Leu-Nap from PAM1723.

Example 4 Mechanism of Efflux Pump Inhibition

The effect of Leu-Nap substrate concentration on inhibition was investigated. For both PAM1626 and PAM1723 strains, change in Leu-Nap fluorescence in 30 min. was plotted as a function of pentamidine concentration for external Leu-Nap concentrations of 120 μg/ml (FIG. 2A) and 60 μg/ml (FIG. 2B). When external concentration of Leu-Nap was 120 μg/ml, 20 μg/ml of pentamidine was required to completely inhibit MexAB-OprM-mediated efflux (i.e., the same fluorescence is produced in PAM1723 and PAM1626). When the Leu-Nap concentration was decreased to 60 μg/ml, 80 μg/ml of pentamidine was required for complete inhibition of the pump. This result implies that the degree of inhibition is inversely dependent on the substrate concentration, indicating uncompetitive inhibition. The effect of the substrate on the degree of inhibition is a strong indication of the efflux pump inhibitory mode of action of pentamidine.

Example 5 Membrane Permeabilization

Both antibiotic potentiation and increased uptake in the presence of pentamidine might be a result of the permeabilization of the outer membrane of P. aeruginosa by this compound. To rule out this possibility a direct outer membrane permeabilization experiment was performed. In this assay the rates of hydrolysis of a chromogenic β-lactam, nitrocefin, by intact cells of P. aeruginosa expressing β-lactamase, was examined. β-lactamase is located in the periplasm. An increased rate of hydrolysis in intact cells is indicative of increased permeation of nitrocefin across the outer membrane since the rate of hydrolysis is limited by the rate of this permeation. The potential outer membrane permeabilizing effect of pentamidine was examined using P. aeruginosa strain PAM2005. This strain constitutively produces β-lactamase AmpC, encoded by the corresponding gene normally present in the genome of this bacterium. PAM2005 also overproduces the MexAB-OprM efflux pump. PMBN, the known outer membrane permeabilizing agent was used for these experiments. Cells were grown overnight in L-broth, harvested, washed in Mg²⁺ free accumulation buffer, and re-suspended in the same buffer at OD₆₀₀ of ˜0.5. To 100 μl of cell suspension, 50 μl of either PMBN or pentamidine was added to give a final concentration ranging from 2 to 64 μg/ml. Next, 50 μl of nitrocefin was added to give a final concentration of 64 μg/ml. Hydrolysis of nitrocefin was monitored spectrophotometrically by measurement of the increase in absorbance at 574 nm. Assays were performed in 96-well plates in a SpectraMAX Plus spectrophotometer (Molecular Devices).

PMBN had a dramatic effect on the permeabilization of the outer membrane with IC₅₀<2 μg/ml (FIG. 3A). In contrast pentamidine had rather weak membrane permeabilization activity with IC₅₀ exceeding 64 μg/ml (FIG. 4). Even this weak activity is completely abrogated when 1 mM Mg²⁺ is added to the reaction buffer (FIG. 4). At this concentration of Mg²⁺, pentamidine has an effect in Leu-Nap accumulation experiments. Thus, it is proposed that pentamidine acts on efflux pumps rather than promoting membrane permeabilization.

Example 6 Proton Gradient Disruption

A proton gradient is necessary for efflux pump activity. Accordingly, compounds that are capable of proton gradient disruption will appear as efflux inhibitors in accumulation assays. This possibility was tested for pentamidine. EtBr is a substrate of the MexAB-OprM efflux pump from P. aeruginosa as evident from the differential rates of its uptake in PAM 1723 and PAM 1626, MexAB-OprM overexpressing or lacking strains, respectively. CCCP is a well-known protonophore, which rapidly dissipates the proton gradient. Treatment of PAM1723 cells with 50 μg/ml of CCCP for 15 minutes resulted in dramatic increase of EtBr uptake (FIG. 5A). In contrast, treatment of cells with pentamidine at 80 μg/ml did not result in increased EtBr uptake (FIG. 5B) indicating that pentamidine unlike CCCP does not disrupt the proton gradient. The pump inhibitory activity of pentamidine is not based on disruption of the proton gradient across the inner membrane of P. aeruginosa.

Example 7 Effect of Pentamidine on Multiple Over-Expressing Efflux Pumps

The impact of fixed concentrations of pentamidine on the susceptibility of fluoroquinolones to various P. aeruginosa strains was determined. Multiple concentrations of antibiotics were tested in the presence of a single chosen concentration of pentamidine. As above, the MIC was defined as the lowest concentration of antibiotic, within the combination, at which the visible growth of the organism was completely inhibited.

A set of strains that simultaneously over-expressed pairs or triplicates of pumps, e.g. PAM2302 and PAM2303 was tested. Simultaneous over-expression of efflux pumps has been recently detected in clinical strains. The data indicate that pentamidine increased susceptibility in strains over-expressing multiple pumps. Moreover, in the presence of pentamidine, susceptibility to both fluoroquinolones was similar.

TABLE 3 Impact of pentamidine on Susceptibility to fluoroquinolones of P. aeruginosa Strains Simultaneously Over-expressing Multiple Efflux Pumps. Levofloxacin MIC (μg/ml) Ciprofloxacin MIC (μg/ml) w/pentamidine w/pentamidine Strain Pump status w/o pentamidine at 20 μg/ml w/o pentamidine at 20 μg/ml PAM1020 none 0.25 0.03 0.06 0.015 PAM1032 MexAB-OprM 2 0.125 0.5 0.06 PAM1033 MexCD-OprJ 4 0.25 1 0.25 PAM1034 MexEF-OprN 4 0.125 1 0.06 PAM1438 MexAB-OprM 4 0.25 16 0.25 MexCD-OprJ PAM2281 MexAB-OprM 8 0.25 8 0.25 MexEF-OprN PAM2282 MexAB-OprM 8 0.25 2 0.125 MexEF-OprN PAM2302 MexAB-OprM 8 0.25 2 0.25 MexCD-OprJ MexEF-OprN PAM2466 MexAB-OprM 2 0.125 0.5 0.06 MexXY-OprM

Example 8 Effect of Pentamidine on Target Mutation Antibiotic Resistance

In addition to increased efflux, resistance to fluoroquinolones may be due to target mutations (gyrase and topoisimerase IV) or due to combination of these two resistance mechanisms in a single bacterial cell. Consequntly, the effect of fixed concentrations of pentamidine on strains of P. aeruginosa containing various combinations of efflux-mediated and target-based mutations was measured. All tested fluoroquinolones were affected by target-based mutations. Moreover, the degree of this impact was similar for all the tested fluoroquinolones in that each mutation demonstrated a 4 to 8-fold effect. As expected, both mechanisms contribute to fluoroquinolone resistance independently. The same contribution of target-based mutations was observed regardless of which pump was over-expressed, and conversely the same contribution of efflux was observed regardless of which target-based mutation was present. Pentamidine increased susceptibility to fluoroquinolones in strains with efflux-based and target-mediated mutations (Table 4).

TABLE 4 Impact of Pentamidine on Susceptibility of P. aeruginosa Strains with Combinations of Efflux and Target Mutations. Levofloxacin MIC (μg/ml) Ciprofloxacin MIC (μg/ml) w/pentamidine w/pentamidine Strain Pump status Target mutation w/o pentamidine at 20 μg/ml w/o pentamidine at 20 μg/ml PAM1020 none none 0.25 0.03 0.06 0.015 PAM1032 MexAB-OprM none 2 0.125 0.5 0.06 PAM1033 MexCD-OprJ none 4 0.25 1 0.25 PAM1034 MexEF-OprN none 4 0.125 1 0.06 PAM1481 MexAB-OprM gyrA (Asp87Tyr) 16 1 4 0.5 PAM1482 MexCD-OprJ gyrA (Asp87Tyr) 16 1 8 2 PAM1483 MexAB-OprM gyrA (Asp87Tyr) 16 1 8 2 MexCD-OprJ PAM1491 MexEF-OprN gyrA (Asp87Tyr) 32 2 8 1 PAM1548 none gyrA (Thr83Ile) 2 0.25 2 0.5 PAM1569 MexCD-OprJ gyrA (Thr83Ile) 32 4 16 4 PAM1570 MexEF-OprN gyrA (Thr83Ile) 32 2 16 2 PAM1573 MexAB-OprM gyrA (Thr83Ile) 16 1 8 1 PAM1582 MexAB-OprM gyrA (Thr83Ile) 64 4 64 8 parC (Ser87Leu) PAM1609 MexAB-OprM gyrA (Thr83Ile) >128 16 >64 16 parC (Ser87Leu) gyrA (Asp87Tyr) PAM1667 none gyrA (Thr83Ile) 8 1 32 4 parC (Ser87Leu) PAM1669 none gyrA (Thr83Ile) 32 4 64 8 parC (Ser87Leu) gyrA (Asp87Tyr)

Example 9 Effect of Pentamidine on P. aeruginosa Isolated from Patients with Cystic Fibrosis

Susceptibility to levofloxacin and ciprofloxacin of 108 recent clinical isolates of P. aeruginosa obtained from cystic fibrosis patients was determined in the presence of fixed concentrations of 20 μg/ml pentamidine. In addition, susceptibilities of the same strains to aminoglycosides tobramycin and gentamicin and beta-lactam aztreonam was determined. These later antibiotics are used to treat P. aeruginosa infections during exacerbation of the disease. Susceptibility of clinical isolates of P. aeruginosa to antibiotics was determined using broth two-fold broth-micro-dilution method in according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. Ciprofloxacin, levofloxacin, and aztreonam were tested at concentrations raging from 64 μg/ml to 0.06 μg/ml. Tobramycin and gentamicin were tested at concentrations raging from 128 μg/ml to 1.25 μg/ml. Additionally, the isolates were tested against ciprofloxacin and levofloxacin in the presence of 20 μg/ml of pentamidine (the same range of fluoroquinolones of 64 μg/ml to 0.06 μg/ml was used). The strains were determined to be Resistant (R), Intermediate (I) or Susceptible (S) according to NCCLS susceptibility breakpoints. Finally, the percent of susceptible organisms was calculated for each antibiotic (FIG. 6). In the case of fluoroquinolone/pentamidine combinations, susceptibility breakpoints were based on the corresponding antibiotics.

The results indicated that among all antibiotics tested, flouroquinolone/pentamidine combinations possessed the most potent anti-pseudomonal activity. This result can be also presented as a distribution of MICs with or without potentiators (FIG. 7). Such presentation allows determination of MIC₅₀ and MIC9₅₀ of the population (i.e., MIC of 50% and 90% of the strains respectively). In the case of this particular panel of strains, pentamidine decreased MIC₅₀ and MIC₉₀ of levofloxacin and ciprofloxacin 8-fold and 2-fold, respectively (Table 5).

TABLE 5 MICs for antibiotics tested. Levo/ Cipro/ Levo pentamidine Cipro pentamidine MIC₅₀ 2 0.25 1 0.125 MIC₉₀ 8 4 8 4

Examples of potentiation of the antibiotic effect by pentamidine against the individual strains are shown in the graph in FIG. 8. The 45 strains shown on this Figure were selected based on their resistance to levofloxacin (MIC>2 μg/ml). Pentamidine decreased levofloxacin MICs of the most of resistant stains 4-fold to 64-fold.

Example 10 Effect of Pentamidine on Bacterial Death

Another way to establish potentiating activity of EPIs is to demonstrate their effect on killing by fluoroquinolones. The strain PAM1032 overexpressing the MexAB-OprM efflux pump was grown in the presence of sub-inhibitory concentration of levofloxacin (½ of MIC or 1 μg/ml) either with no addition or in the presence of 10 μg/ml or 20 μg/ml of pentamidine. The effect of pentamidine on killing was compared to the effect of 4 μg/ml of levofloxacin (2×MIC). While pentamidine alone did not have any effect on killing of P. aeruginosa (FIG. 9A), it significantly inhanced levofloxacin killing (FIG. 9B). In fact, 10 μg/ml of pentamidin in the presence of ½×MIC of levofloxacin had similar effect on killing of P. aeruginosa as 2×MIC and 20 had even stronger effect.

Example 11 Efflux Pump Inhibitory Activity of Pentamidine Analogs

Efflux pump inhibitory activity of three pentamidine analogs were evaluated using the levofloxacin potentiation checkerboard assay against five strains of P. aeruginosa overexpressing or lacking efflux pumps (as in Example 1). All three compounds, propamidine, dibromopropamidine, and hexamidine potentiated levofloxacin against the strains overexpressing various efflux pumps. The levofloxacin potentiating activity of these compounds was comparable to pentamidine. For example, all three compounds and pentamidine reduced the levofloxacin MIC of the strain PAM1723 (2 μg/ml) overexpressing the MexAB-OprM efflux pump at least 8-fold at concentrations of 10 μg/ml to 20 μg/ml (Table 5). Importantly, the MICs of all tested pentamidine analogs against PAM1626 lacking efflux pumps were lower than for the strains expressing efflux pumps. This indicates that similar to pentamidine, pentamidine analogs are substrates of these pumps.

TABLE 5 Potentiation of levofloxacin by several pentamidine analogs against the strains of P. aeruginosa expressing or lacking various efflux pumps. Pump which Levofloxacin MIC (μg/ml) in the presence of contributes to MIC* propamidine [MP 001039] (μg/ml) Strain Relevant genotype resistance (μg/ml) 0 1.25 2.5 5 10 20 40 80 A. Propamidine PAM1020 wt MexAB-OprM 40 0.25 0.25 0.125 0.125 0.06 0.03 NG NG PAM1723 nalB ΔmexCD-oprJ MexAB-OprM 40 2 2 2 1 0.5 0.25 NG NG ΔmexEF-OprN PAM1738 nfxB ΔmexCD-oprJ MexCD-OprJ 40 0.5 0.5 0.5 0.25 0.125 0.015 NG NG ΔmexAB-OprM PAM1753 nfxC ΔmexAB-oprM MexEF-OprN 5 4 4 2 NG NG NG NG NG ΔmexCD-OprJ PAM1626 ΔmexAB-OprM none 10 0.015 0.015 0.007 0.007 NG NG NG NG ΔmexCD-oprJ ΔmexEF-OprN

Pump which Levofloxacin MIC (μg/ml) in the presence of contributes to MIC* dibromopropamidine [MP 001040] (μg/ml) Strain Relevant genotype resistance (μg/ml) 0 1.25 2.5 5 10 20 40 80 B. Dibromopropamidine PAM1020 wt MexAB-OprM 80 0.25 0.25 0.25 0.125 0.06 0.03 0.03 NG PAM1723 nalB ΔmexCD-oprJ MexAB-OprM 80 2 2 1 0.5 0.125 0.06 0.06 NG ΔmexEF-OprN PAM1738 nfxB ΔmexCD-oprJ MexCD-OprJ 80 0.5 0.5 0.25 0.125 0.06 0.03 0.03 NG ΔmexAB-OprM PAM1753 nfxC ΔmexAB-oprM MexEF-OprN 20 4 2 1 0.5 0.125 NG NG NG ΔmexCD-OprJ PAM1626 ΔmexAB-OprM none 5 0.015 0.0075 0.007 NG NG NG NG NG ΔmexCD-oprJ ΔmexEF-OprN

Pump which Levofloxacin MIC (μg/ml) in the presence of contributes to MIC* hexamidine [MP 001041] (μg/ml) Strain Relevant genotype resistance (μg/ml) 0 1.25 2.5 5 10 20 40 80 C. Hexamidine PAM1020 wt MexAB-OprM 20 0.25 0.125 0.125 0.03 0.007 NG NG NG PAM1723 nalB ΔmexCD-oprJ MexAB-OprM 40 2 2 1 1 0.125 0.015 NG NG ΔmexEF-OprN PAM1738 nfxB ΔmexCD-oprJ MexCD-OprJ 80 0.5 0.5 0.5 0.25 0.06 0.015 0.007 NG ΔmexAB-OprM PAM1753 nfxC ΔmexAB-oprM MexEF-OprN 20 4 2 2 1 0.25 NG NG NG ΔmexCD-OprJ PAM1626 ΔmexAB-OprM none 2.5 0.015 0.007 NG NG NG NG NG NG ΔmexCD-oprJ ΔmexEF-OprN

Example 12 Accumulation Assays of Pentamidine Analogs

Efflux pump inhibitory activity of propamidine, dibromopropamidine, and hexamidine was confirmed in Leu-Nap accumulation assays. The uptake of Leu-Nap (100 μg/ml) by PAM1723 (FIGS. 10A, C, and E) or PAM1626 (FIGS. 10B, D, and F) cells was studied in the presence of various concentrations of propamidine (0 μg/ml to 160 μg/ml), dibromopropamidine (0 to μg/ml to 40 μg/ml), and hexamidine (0 μg/ml to 40 μg/ml), respectively. All three compounds were capable of completely inhibiting the MexAB-OprM-mediated efflux of Leu-Nap from the strain overexpressing this pump. The rate of Leu-NAp uptake into PAM1626, and PAM1723, in the presence of 160 μg/ml propamidine, 20 μg/ml of dibromoproapmidine, and 20 μg/ml of hexamidine was the same.

Example 13 Efflux Pump Inhibitory Activity of Diamidine Analogs

Efflux pump inhibitory activity of several commercially available diamidine analogs was evaluated using the checkerboard assay of Example 1 against the strain of P. aeruginosa overexpressing the MexAB-OprM efflux pump. Two compounds with measurable efflux pump inhibitory activity are shown in Table 6.

TABLE 6 Potentiation of levofloxacin by diamidine analogs relative to pentamidine against the strain of P. aeruginosa, PAM1723, expressing the MexAB-OprM efflux pump. MIC* Levofloxacin MIC (μg/ml) in the presence of dimidines (μg/ml) Compound (μg/ml) 0 1.25 2.5 5 10 20 40 80 Pentamidine MP 601205

>81 2 2 1 1 1 0.25 0.06 0.007 MP 601206

80 2 2 2 1 1 0.5 0.06 NG MP 601319

>80 2 2 2 2 2 1 0.25 0.125

Example 14 Aerosolized Delivery of Pentamidine (Particle Size Optimization)

To ascertain the safety and tolerability of pentamidine delivered to subjects with stable cystic fibrosis, a 10 subject Phase Ib study was performed. In addition to safety and tolerability, pentamidine pharmacokinetic observations were also made in sputum, plasma and urine. In this study, subjects were administered four total doses of aerosolized Nebupent (the FDA-approved form of pentamidine isethionate) using both the FDA-indicated Respirgard II jet nebulizer (1.7 micron VMD particle, or ˜1 micron MMAD) and an Aerogen Clinical vibrating mesh device (3.4 micron VMD particle, or ˜2 micron MMAD). Doses for each device were determined in vitro and adjusted to deposit both a high dose (predicted equivalent depostited dose from administration of the FDA-approved dose in the Respirgard II device) and ⅓ of the high dose. (Predicted deposted dose was calculated from in vitro models that take into account the particle size distribution of drug and the efficiency of device delivery—see Examples 16 and 17). The predicted high and low deposited doses were ˜20 mg and ˜7 mg pentamidine, respectively. For the Respirgard II device the high deposited dose was acquired from a dose of 300 mg/6 ml and low deposited dose from 100 mg/6 ml. By comparison, the Aerogen device high deposited dose was acquired from 70 mg/4 ml and low deposited dose from 25 mg/4 ml. Each subject was randomized to receive the low dose using either the Respirgard II or Aerogen device first, then following a seven-day wash out period, received the high dose using the same device. After a second seven-day wash out period, subjects again received the low dose, but with the other device. Finally, after a third seven-day washout, subjects received their final administration, a high dose using the same second device.

TABLE 7 Device Performance and Dose Selection Parameter Aerogen^(A) (Low) Aerogen^(A) (High) Respirgard (Low) Respirgard (High) NebuPent Soln. 25 mg/4 ml H₂0 70 mg/4 ml H₂0 100 mg/6 ml H₂0 300 mg/6 ml H₂0 VMD (GSD) 3.4 (1.9) micron 3.4 (1.9) micron 1.7 (2.4) micron 1.7 (2.4) micron Inhaled Mass 8.6 mg 24.5 mg 6.7 mg 20.1 mg Deposited Dose^(B) 6.9 mg 19.7 mg 6.4 mg 19.3 mg ^(A)Aerogen Clinical device is a modification of the Aeroneb Go in that it contains a smaller particle size and contains an expiration filter. ^(B)Predicted deposted dose calculated from in vitro models that take into account the particle size distribution of drug and the efficiency of device delivery. Specifically, using a normal tidal flow breath simulator, the inhaled mass was multiplied by the fine particle fraction (% particles ≦5 micron VMD).

The pharmacokinetic data generated in the study indicated that systemic absorption of pentamidine was insignificant. Of the 96 plasma samples analyzed, only four showed levels of pentamidine above the lower limit of quantitation (LLQ=0.015 μg/mL). The maximum value found was 0.04 μg/mL and no quantifiable levels were noted later than 30 minutes after administration. Urinary levels were higher than plasma levels, with approximately half the urine samples showing values above the LLQ, but the maximum level noted was 0.33 μg/mL and the values were sporadic enough that further pharmacokinetic analysis was judged unlikely to yield significant information. Low systemic exposure was expected for the Respirgard (˜1 μm particle size). However, there was concern that a larger particle size would increase systemic exposure. This event was not observed with the Aerogen device (˜2 μm particle).

Delivery of pentamidine in sputum was superior for the Aerogen device, as shown by significantly higher Cmax and AUC, even though the respirable dose for both devices was calibrated by in vitro evaluation to be equal (Table 8). Cmax for the Aerogen high-dose was 15.9 μg/mL compared to 6.4 μg/mL for the Respirgard high dose. The α phase half-life for elimination from sputum was similar (ca. 2.2-2.6 hours) with both devices. Twelve of thirteen measured samples had quantifiable levels of pentamidine on Day 7 (≦0.11 μg/ml). The inter-subject pentamidine sputum half-lives varied considerably, although the highest measured concentration generally occurred at the first analyzed time point (1 hour). The majority of subjects had a calculated high-dose β-phase elimination half-life between one and two days. The sputum pharmacokinetic data suggest that pentamidine concentrations in the lung are in a viable range for efficacy, but a 2-fold increase in AUC is desirable. The biphasic elimination kinetics suggest that some drug accumulation may occur upon multi-day dosing, advantageously increasing AUC levels.

TABLE 8 CF Sputum pharmacokinetic results AUC (0-4)/ AUC (0-8)/ AUC (0-24)/ AUC AUC AUC AUC (0 to AUC (0 to last AUC (0 to AUC (0 to 4) AUC (0 to 8) AUC (0 to 24) (0-168) (0-168) (0-168) 168) non-BLQ) infinity) μg/mL * hours μg/mL * hours μg/mL * hours % % % μg/mL * hours μg/mL * hours μg/mL * hours Aerogen Clinical Mean 33 46 63 46 61 77 84 83 84 SD 20 26 35 27 27 21 48 48 49 Respirgard II Mean 12 17 31 30 41 66 57 56 57 SD 12 13 24 16 18 17 66 66 67 T_(max) (0 to 168) C_(max) (0 to 168) K_(e) (a)^(A) T_(1/2) (a)^(A) K_(e) (β)^(A) T_(1/2) (β)^(A) (hours) (μg/mL) (1/hours) (hours) (1/hours) (hours) Aerogen Clinical Mean 1.8 15.9 0.364 2.2 0.070 24.4 SD 1.4 12.5 0.151 0.8 0.085 15.3 Respirgard II Mean 2.3 6.4 0.326 2.6 0.074 26.7 SD 2.5 6.3 0.149 1.4 0.128 16.5 ^(A)The a phase was 0-8 hours (0, 1, 4, and 8 hours) and the β phase was based on the best-fitted line that included the last measurable value.

Without being bound to any particular theory, it is believed that the increase in particle size of the Aerogen device may explain the increase in sputum deposition. Previous clinical studies measuring the affects of pentamidine in HIV patients and healthy volunteers showed increased coughing with significantly larger particle sizes (<5 micron MMAD). However, with other aerosol antibiotics, an intermediate particle size (3-4 micron MMAD) results in higher CF sputum drug concentrations. Thus, a device emitting an intermediate particle size and exhibiting a small geometric standard deviation has the potential to significantly increase sputum drug levels while maintaining tolerability.

Based upon these results, increasing the aerosol particle size with the Aerogen Clinical device (and exhibiting a small GSD, e.g. less than 1.9 micron) improved pentamidine deposition and related pharmacokinetic properties. Moreover, this particle size is not expected to increase the risk to subjects receiving these single doses of pentamidine due to lack of systemic administration.

Aerosolized pentamidine tolerability can be improved by optimizing the osmolarity, permeant ion content, and/or substituting salts to reduce coughing/bronchospasm, as well as a change in excipients to improve taste. TOBI is a good example of the opportunity/feasibility for improving NebuPent. Early studies aerosolizing IV tobramycin was associated with significant coughing and resulted in several incidences of bronchospasm. It was found that both osmolarity and chloride content correlated with the incidence of bronchospasm. Learning from these studies, IV tobramycin was reformulated (TOBI) to maintain the osmolarity between 150 and 200 mOsmol/kg, chloride-content approximately one-half that of saline, and a pH of 6. Further, specifically targeting aerosol deposition to the middle airways (˜3.5 micron MMAD), TOBI has been shown to be well-tolerated and efficacious in CF patients. Thus, by modifying these physico-chemical and device related factors, poorly tolerated IV tobramycin was successfully reformulated and marketed with high patient compliance.

NebuPent exhibits negative acute respiratory side affects in HIV patients similar to that observed in the early experience with nebulized IV tobramycin. Although common, these side-effects are usually controllable without discontinuing treatment. As discussed, particle size and deposited dose have been implicated as factors contributing to NebuPent-induced cough and bronchospasm, which is largely determined by device. However, formulation must also be considered as a significant contributor. Similar to aerosolized tobramycin before TOBI, NebuPent is a simple conversion of the IV pentamidine formulation for aerosolization. Because of the clinical urgency for an anti-Penumocystis agent, formulation was skipped entirely for NebuPent and IV pentamidine was only optimized for particle size in order to achieve deposition in the alveolar spaces (site of efficacy for PCP).

NebuPent only consists of pentamidine and its counter-ion salt, isethionate. This substance is manufactured as a lyophilized package to provide stability for water reconstitution immediately before use. In comparison to TOBI, at concentrations used in this clinical study (near efficacious concentrations) the osmolarity of NebuPent is ˜95 mOsmol/kg, is devoid of chloride, and has a pH of 4.9. Given what is known about the relationship between these factors and tolerability (optimum pH between 5.5-7, osmolarity between 150-550 mOsm/kg, permeant ion concentration between 31-300 mM), these observations suggest that in addition to device optimization, the EPI pentamidine should be reformulated to increase pH, osmolarity and chloride content. Further, because the thio groups of the isethionate salt of pentamidine have been suggested as an airway irritant, changing the pentamidine salt may further decrease the incidence of acute intolerability.

Example 15 Aerosolized Delivery of Pentamidine and Pentamidine/Levofloxacin (Optimizing Solution Formulation)

In a single subject, healthy volunteer study, NebuPent (the FDA-approved form of pentamidine isethionate) was administered using either the Aerogen Clinic nebulizer used in Example 14 (Aerogen Small), or an Aerogen Clinic nebulizer (Aerogen Large) similar in all respects except for pore size, resulting in a larger particle size of ˜4.5 micron MMAD. NebuPent was administered and tested at different concentrations of saline as well as a fixed combination trial where NebuPent and Levaquin (sterile preservative-free levofloxacin in 500 mg/20 mL vials in water, pH adjusted) were mixed and co-aerosolized. 1× (Normal) saline is 0.85% NaCl.

In the first trial, NebuPent was dissolved in distilled water to a concentration of 18.75 mg/ml and inhaled using either the Aerogen Small or Aerogen Large nebulizer. Delivery of NebuPent in the Aerogen Small device was better tolerated than in the Aerogen Large device. With the Aerogen Small device, there was a slight cough sensation and poor taste experienced during the later part of the administration. Further, slight cough and clearing of the throat was present for a short period after administration. With the Aerogen Large device, cough sensation and throat irritation occurred starting earlier during drug administration and transient coughing occurred during the later stage of administration, and for a period of time thereafter. Results from this trial indicate that NebuPent administered in a smaller particle size device (˜2 micron MMAD, ˜2.0 micron GSD) is better tolerated than in a larger particle size device (˜3.7 micron MMAD, ˜2.0 micron GSD) that produces a significant fraction of particles greater than 5 micron MMAD.

In the second trial, 18.75 mg NebuPent was dissolved in either 1 ml distilled water or 1 ml 0.425% NaCi (0.5× saline). These solutions of NebuPent were then inhaled using the Aerogen Large device. The 0.5× saline dissolved NebuPent demonstrated a clear tolerability improvement over NebuPent dissolved in distilled water. Qualitatively, the 0.5× saline solution induced a delayed mild throat irritation that stimulated a single cough over the administration period. By comparison, in the absence of NaCl, there was an immediate, intense throat irritation that resulted in transient cough at the end of dose, similar in experience to the results for the Aerogen Large device in the first trial. The greatest difference was noted following completion of dose administration, in that the solution lacking NaCl resulted in throat-clearing and moderate cough after administration.

In the third trial administration of NebuPent with the Aerogen Large nebulizer, 0.5× saline was compared to NebuPent in 1× saline. The tolerability of the 1× saline solution was noticeably better than the NebuPent in 1× saline solution. Qualitatively, the 0.5× saline solution was similar to the experience for the 0.5× saline solution observed in the second trial. There was a very slight chemical taste during the first part of the administration, which became more noticeable in the later part of the administration. A slight coughing sensation was also noticeable in the later part of the dosing, followed by a short period after administration where clearing of the throat occurred. With the 1× saline solution, there was no noticeable taste or cough sensation throughout the entire administration period, followed by a slight taste noticeable for a brief period immediately after administration. There was no coughing or throat clearing during or after administration.

In the fourth trial, 18.75 mg NebuPent was dissolved in 1 ml distilled water (no saline) in the presence of 18.75 mg levofloxacin HCl (from Levaquin). For the comparison purposes, this solution of NebuPent/levofloxacin was again inhaled using the Aerogen Large device. Results from this trial indicate that this NebuPent/levofloxacin mixture was similarly tolerated with NebuPent in 1× saline.

In the fifth trial, 18.75 mg of NebuPent was prepared with 18.75 mg levofloxacin HCL (from Levaquin) in a 1 ml volume to a final saline concentration of 0.25×, and administered in the Aerogen Lagre device. The tolerability of this pentamidine/levofloxacin combination-saline solution was similarly tolerated as the pentamidine-levofloxacin solution tested in trial four, and similarly tolerated to to the pentamidine 1× saline solution tested in trial three.

These results suggest that either or both osmolarity and chloride content contribute to the improved tolerability of NebuPent aerosol administration. The contribution of each parameter is shown in the Table 9. These data support an ideal osmolarity within the range of 150-550 mOsm/kg and an ideal permeant ion content within the range of 31-300 mM.

TABLE 9 Osmolarity and Chloride content of Tested Pentamidine Formulations Osmolarity: 0.5X Saline 154 mOsm/kg 18.75 mg/ml NebuPent 95 mOsm/kg 18.75 mg/ml NebuPent in 0.5X saline 249 mOsm/kg 18.75 mg/ml NebuPent in 1X saline 403 mOsm/kg 18.75 mg/ml Levaquin 50 mOsm/kg + approx. 50 mOsm HCL/NaOH 18.75 mg/ml NebuPent/Levaquin 145 mOsm/kg + approx. 50 mOsm HCL/NaOH Chloride Content: 0.5X Saline 154 mM 18.75 mg/ml NebuPent 0 mM 18.75 mg/ml NebuPent in 0.5X saline 154 mM 18.75 mg/ml NebuPent in 1X saline 308 mM 18.75 mg/ml Levaquin approx. 25 mM 18.75 mg/ml NebuPent/Levaquin approx. 25 mM

Example 16 Aerosol Particle Size Determination

Particle size determination of emitted aerosol of an efflux pump inhibitor disclosed herein is conducted with a Malvern Spraytec particle sizer under the following conditions. Ambient conditions are controlled to maintain a room temperature of between 23.0° and 24.0° and relative humidity of 42% to 45%. The efflux pump inhibitor is reconstituted in water or other formulation described herein for loading the nebulizer of choice. Concentrations for particle sizing are between 1 and 100 mg/ml. Software for the Malvern Spraytec particle sizer is programmed to calculate the following information. A) Volume Mean Diameter (VMD), the volume mean of the particles passing across the beam of the laser. B) Geometric Standard Deviation (GSD), diameter 84^(th) percentile/diameter 50^(th) percentile. C) % of particles ≦5μ, the percent of the number of particles less than 5 microns or % of particle >1μ and <7μ, the percent of the number of particles between 1 and 7 microns.

The device is loaded with 2 ml of reconstituted drug. The mouthpiece of the device is positioned with the tip of the mouthpiece 2 cm from the center of the beam on the x axis and as close to the optical lens of the laser as possible on the y axis. Ambient conditioned, bias flow is provided through the nebulizer in an amount to obtain a total nebulizer flow of 20 LPM. By non-limiting example this flow is 13 liters per minute (LPM) for the Respirgard II and 20 LPM for the Aeron Clinical. The nebulizer is turned on (for example, 7 LPM compressed dry medical grade air for the Respirgard II) and allowed to run continuously for 1 minute prior to measuring. The measurement sequence is begun after 1 minute and measurements are made continuously for 1 minute in 1 second intervals (total of 60 records). At the end of the measurement phase, these 60 records are averaged for VMD, GSD and % ≦5 micron and % >1 and <7 micron. Finally, the nebulizer is weighed for determination of output rate.

Example 17 Determination of Respirable Delivered Dose

The respirable delivered dose delivered by an aerosol producing device is measured under conditions similar to natural inhalation using a breath simulator apparatus. By non-limiting example, such a breath simulator uses the PARI Compas breath Simulator programmed to use the European Standard pattern of 15 breaths per minute with an inspiration to expiration ratio of 1:1. Such measurements are performed under ambient conditions that can be controlled to maintain a room temperature of between 23.0° and 24.0° and relative humidity of 42% to 45%. The efflux pump inhibitor is reconstituted in water or other formulation described herein for loading the nebulizer of choice. Concentrations for breath simulation experiments are between 1 and 100 mg/ml.

By non-limiting example, the Respirgard II or Aerogen Clinical devices are loaded with 300 mg pentamidine isethionate in 6 ml or 70 mg in 4 ml water or other formulation described herein, respectively. Breathing simulation is commenced, and the nebulizers activated. The Respirgard II device is powered by 7 LPM of dry, compressed, medical grade air. The devices are allowed to run continuously until to the onset of sputter (in the Respirgard II) or until nebulization ceases (as in the Aerogen Clinical). The duration is timed from the beginning of nebulization. Following nebulization, the inspiratory and expiratory filters are individually washed in a known amount of solvent (dH₂O). The nebulizer cup is also washed individually. For the Respirgard device, only the nebulizer cup itself is rinsed and the drug contained therein assayed. For quantitation, the individual washings are assayed via spectrophotometry at a wavelength of 263 nanometers and the resultant concentration converted to content. Using this quantitative data, the following analysis are made. A) Inspired dose (ID), the total amount of drug assayed from the inspiratory filter. B) Residual dose (RD), the amount of drug assayed from the nebulizer at the end of nebulization. C) Fine Particle Dose (FPD), the ID multiplied by the respirable fraction (for example, % particles ≦5 microns VMD or MMAD depending on the method used to determine the size of the particles emitted from the selected device). D) Duration, time from the beginning to the end of nebulization. E) FPD/min, the FPD divided by the duration. F) Delivered Dose %, the percent device-loaded drug dose that is captured on the inspiration filter. G) Respirable Delivered Dose, % ID that is, for example, ≦5 microns VMD or MMAD. The following data is obtained:

Residual Insp. Delivered Duration Dose Dose FPD/min FPD VMD GSD Dose % Respirgard 32.6 ± 1  135 ± 10 20.9 ± 2 0.62 ± .01 20.1 ± 2   1.7 2.4  6.7% Aerogen 20.2 ± 2  4.2 ± 2  29.9 ± 2  1.2 ± 0.2 24.1 ± 1.8 3.4 1.9 34.5% Clinical Respirable Respirable FPD delivered delivered (1-7μ) % ≦ 5μ % > 1 < 7μ dose (≦5μ) dose (1-7μ) Respirgard 11.1 ± 1.3   96%   53% 19.2 10.6 Aerogen 22.1 ± 1.9 80.4% 73.6% 19.4 17.8 Clinical

Example 18 Clinical Treatment of Antibioitic Resistance Infections

Pentamidine or any of the other efflux pump inhibitor compounds disclosed herein is coadministered with a conventional antibiotic to treat a patient suffering from a gram-negative bacterial infection, wherein the bacteria are resistant via the mechanism of efflux pump mediated multidrug resistance. The dosage of the antibiotic is from 20%, 30%, 40%, or 50% up to 70%, 80%, 90%, or 100% of the established therapeutic dosage. The antibiotic is administered orally, IV, IP, IM, or via inhalation, and the efflux pump inhibitor is administered to the lungs via inhalation of 300 mg dosage of 2-4 νm aerosol droplets.

All references cited herein are incorporated herein by reference in their entirety. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. It is intended that the specification and examples be considered as exemplary only. 

1. A method of therapeutic treatment of a gram negative bacterial infection in a subject, comprising co-administering to a subject infected with a gram negative bacteria having an efflux pump an effective amount of an antimicrobial agent an a compound of formula I:

wherein R₁ and R₂ are separately selected from the group consisting of amine and C₁ ₋ ₄ alkyl amine; Linkers L1 and L3 are separately selected from the group consisting of amine and alkylamine or are separately absent; aromatic rings A₁ and A₂ are separately selected from the group consisting of

wherein Z₁-Z₄ C, with the proviso that aromaticity of the aromatic rings are maintained; Z₁-Z₄ that are C are optionally substituted with C₁₋₄ alkyl, CH₂NH₂, halogen, methoxy, CH₂C(O)NMe₂, C(O)NH₂, C(O)NMe₂, SO₂Me, or SO₂NH₂; linker L₂ is a 1 to 12 unit chain wherein each unit is independently selected from the group consisting of CH₂, C(CH₃)₂, O, C(O), S, S(O), S(O)₂, NH, NR₄, ═N—, phenyl, monocyclic 5-membered heteroaryl, monocyclic 6-membered heteroaryl, —CH═CH— cis, —CH═CH— trans, NHC(O)NH, NR₄C(O)NH, NHC(O)NR₄, NR₄C(O)NR₄, OC(O)NH, NR₄C(O)O, OC(O)NR₄, and NHC(O)O with the proviso that L₂ does not contain a C(O)NH, C(O)NR₄, C(O)O, or C(O)S unit; wherein the 5-membered heteroaryls are selected from the group consisting of imidazole, furane, thiophene, thiazole, isothiazole, oxazole, isoxazole, 1,2,3-oxadiazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, 1,2,3-triazole, and 1,3,4-triazole; the 6-membered heteroaryls are selected from the group consisting of pyridine, pyrimidine, pyridazine, 1,2,4-triazine, and 1,3,5-triazine; and R₄ is selected from the group consisting of H and C₁₋₄ alkyl.
 2. The method of claim 1, further comprising identifying the subject as a subject infected with a gram negative bacteria that is resistant to the antimicrobial agent.
 3. The method of claim 1, further comprising identifying the subject as a subject infected with a gram negative bacteria that is capable of developing resistance to the antimicrobial agent.
 4. The method of claim 2 or 3, wherein the resistance is at least partly efflux pump-mediated.
 5. The method of claim 4, wherein an efflux pump inhibitory concentration of the compound of formula (I) sufficient to overcome or suppress the emergence of efflux pump-mediated resistance in said bacteria is administered.
 6. The method of claim 1, wherein R₁ and R₂ are amine groups.
 7. The method of claim 1, wherein L₂ is a 1 to 12 unit chain containing units selected from the group consisting of —CH₂—, —C(CH₃)₂—, —O—, —C(O)—, —S—, —S(O)—, —CH═CH— cis, and —CH═CH— trans.
 8. The method of claim 1, wherein the compound of formula I is selected from the group consisting of pentamidine, propamidine, hexamidine, dibromopropamidine,


9. The method of claim 1, wherein an effective efflux pump inhibitory concentration of the compound of formula I remains at the site of infection for at least about a 2 hour period.
 10. The method of claim 1, wherein an effective efflux pump inhibitory concentration of the compound of formula I remains at the site of infection for at least about 4 hour period.
 11. The method of claim 1, wherein an effective efflux pump inhibitory concentration of the compound of formula I is at the site of infection at a same time as the antimicrobial agent is present at its peak period.
 12. The method of claim 1, wherein an effective efflux pump inhibitory concentration of the compound of formula I is at the site of infection for at least about 25% of the time the antimicrobial agent is present at its peak period.
 13. The method of claim 1, wherein an effective efflux pump inhibitory concentration of the compound of formula (I) sufficient to improve an AUC/MIC ratio of the antimicrobial agent by at least about 25% is administered.
 14. The method of claim 1, wherein an effective efflux pump inhibitory concentration of the compound of formula (I) sufficient to improve time-above-MIC for the antimicrobial agent by at least about 25% is administered.
 15. The method of claim 1, wherein an effective efflux pump inhibitory concentration of the compound of formula (I) sufficient to cause a therapeutic effect is administered.
 16. The method of claim 1, wherein the site of infection is localized.
 17. The method of claim 16, wherein the site of infection is a pulmonary site.
 18. The method of claim 17, wherein the bacteria is a Pseudomonas.
 19. The method of claim 17, wherein the compound of formula I is administered as an aerosol.
 20. The method of claim 19, wherein the compound of formula I is administered in one or more administrations so as to achieve a respirable delivered dose daily of at least about 15 mg.
 21. The method of claim 19, wherein the compound of formula I is administered in one or more administrations so as to achieve a respirable delivered dose daily of at least about 40 mg.
 22. The method of claim 19, wherein the compound of formula I is administered in one or more administrations so as to achieve a respirable delivered dose daily of at least about 60 mg.
 23. The method of claim 1, wherein said administering step comprises administering said antimicrobial agent and the compound of formula I by separate routes of administration.
 24. The method of claim 1, wherein said antimicrobial agent and the compound of formula I are administered sequentially.
 25. The method of claim 1, wherein said antimicrobial agent and the compound of formula I are administered simultaneously.
 26. The method of claim 1, wherein said antimicrobial agent and the compound of formula I are administered in a combined, fixed dosage form.
 27. The method of claim 1, wherein said antimicrobial agent is a substrate of an efflux pump in said bacteria.
 28. The method of claim 1, wherein the antimicrobial agent is an antibacterial agent.
 29. The method of claim 1, wherein the antimicrobial agent is a quinolone.
 30. The method of claim 29, wherein the antimicrobial agent is a fluoroquinolone.
 31. The method of claim 30, wherein the antimicrobial agent is selected from the group consisting of ciprofloxacin, levofloxacin, moxifloxacin, ofloxacin, gatifloxacin, cinoxacin, gemifloxacin, and norfloxacin, lomofloxacin, pefloxacin, garenoxacin, sitafloxacin, DX-619.
 32. The method of claim 1, wherein the antimicrobial agent is selected from the group consisting of an aminoglycoside, beta-lactam, coumermycin, chloramphenical, lipopeptide, glycopeptide, glycylcycline, ketolide, macrolide, oxazolidonone, rifamycin, streptogramin, and tetracycline.
 33. The method of claim 1, wherein the bacteria is selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, and Bacteroides splanchnicus.
 34. The method of claim 1, wherein said subject is a human.
 35. The method of claim 1, wherein said subject is a human with cystic fibrosis.
 36. The method of claim 1, wherein said subject is a human with pneumonia, a chronic obstructive pulmonary disease, or sinusitis, or a human being mechanically ventilatated.
 37. A method for reducing the onset of a gram negative bacterial infection in a subject, comprising co-administering to a subject at risk of infecting with a gram negative bacteria having an efflux pump an effective amount of an antimicrobial agent an a compound of formula I:

wherein R₁ and R₂ are separately selected from the group consisting of amine and C₁₋₄ alkyl amine; Linkers L1 and L3 are separately selected from the group consisting of amine and alkylamine or are separately absent; aromatic rings A₁ and A₂ are separately selected from the group consisting of

wherein Z₁-Z₄ are C, with the proviso that aromaticity of the aromatic rings are maintained; Z₁-Z₄ that are C are optionally substituted with C₁₋₄ alkyl, CH₂NH₂, halogen, methoxy, CH₂C(O)NMe₂, C(O)NH₂, C(O)NMe₂, SO₂Me, or SO₂NH₂; linker L₂ is a 1 to 12 unit chain, wherein each unit is independently selected from the group consisting of CH₂, C(CH₃)₂, O, C(O), S, S(O), S(O)₂, NH, NR₄, ═N—, phenyl, monocyclic 5-membered heteroaryl, monocyclic 6-membered heteroaryl, —CH═CH— cis, —CH═CH— trans, NHC(O)NH, NR₄C(O)NH, NHC(O)NR₄, NR₄C(O)NR₄,OC(O)NH, NR₄C(O)O, OC(O)NR₄, and NHC(O)O with the proviso that L₂ does not contain a C(O)NH, C(O)NR₄, C(O)O, or C(O)S unit; wherein the 5-membered heteroaryls are selected from the group consisting of imidazole, furane, thiophene, thiazole, isothiazole, oxazole, isoxazole, 1,2,3-oxadiazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, 1,2,3-triazole, and 1,3,4-triazole; the 6-membered heteroaryls are selected from the group consisting of pyridine, pyrimidine, pyridazine, 1,2,4-triazine, and 1,3,5-triazine; and R₄ is selected from the group consisting of H and C₁₋₄ alkyl.
 38. The method of claim 37, further comprising identifying the subject as a subject at risk of said bacterial infection. 